The role of gut microbiota and chronic inflammation as drivers of cardiovascular disease - Nijmegen Biomedical Study - Non Invasive markers of Atherosclerosis 3

There is overwhelming evidence that chronic inflammation plays a critical role
in both metabolic and cardiovascular diseases (CVD). Recent evidence points at
a crucial role for the commensal gut microbiota. The composition of the gut
microbiota is determined by dietary, environmental and host genetic factors. It
produces a vast amount of metabolites that control host inflammatory responses
in the adipose tissue, liver and blood leukocytes. In turn, these organs, in
combination with the gut metabolites, determine the susceptibility to
developing CVD. Besides metabolic processes, leakage through the intestinal
barrier can also lead to bacterial components entering into the systemic
circulation and activating the
host immune system. We hypothesize that the activation of chronic inflammation
is initiated and driven by a disturbed interaction between gut microbiota, the
intestine, liver and adipose tissue. The resulting chronic inflammation
increases the risk for atherosclerosis and cardiovascular disease.

- To identify the causes of inflammation of adipose tissue, liver and blood
leukocytes by focusing on the contribution of the gut microbiota taking into
account dietary habits and genetic background.
- To assess the influence of inflammation of adipose tissue, liver and blood
leukocytes on non invasive markers of atherosclerosis (NIMA) and CVD risk.

A prospective case-control study will be performed in the Radboud University
Medical Centre (RUMC). The duration of the study is 5 years. This study is an
extension of a large population based cohort study on the relation between
non-invasive measurements of atherosclerosis and cardiovascular risk: the
Nijmegen Biomedical Study - Non Invasive Measures of Atherosclerosis 2
(NBS-NIMA 2). 500 of the original 1,517 participants will be invited to
participate. We will use several approaches to investigate the above described
factors:

a. Baseline characteristics will be collected from all the participants using
questionnaires. These questionnaires will be coded with full respect for the
privacy of the individuals, will be anonymous, and will not permit the
identification of the individuals.
b. Microbiome analysis will be performed on stool, oral, and skin samples.
c. The effect of gut microbiota on the metabolic profile of subjects will be
investigated by conventional laboratory tests. Mass spectrometry,
chromatography, and nuclear magnetic resonance (NMR) spectroscopy will be
performed in collaboration with the other participants of the research
consortium to assess the metabolome.
d. The function of the immune system will be analysed at several levels using
circulating cells from venous blood: immunophenotyping will be done using
fluorescence activated cell sorting (FACS) analysis, circulating factors will
be measured in plasma or serum, in-vitro stimulations of cells and analysis of
mRNA and cytokine responses.
e. In vitro studies will be performed on adipocytes acquired by biopsy of
subcutaneous abdominal and thigh adipose tissue. These will be characterized
for adipocyte functionality and the type and degree of inflammation, with a
focus on two crucial processes for AT inflammation: activation of the
inflammasome and modulation by autophagy.
f. Fat mass and fat distribution will be assessed using anthropometric
measurements, magnetic resonance imaging (MRI) of the trunk and thigh region,
MR spectroscopy of the liver.
g. Subclinical atherosclerosis will be investigated by ultrasonography of the
carotid arteries, pulse wave velocity (PWV), pulse wave analysis (PWA) and
ankle-brachial index (ABI); furthermore subjects are followed for 3-5 years to
register incident cardiovascular diseases (CVD).

Burden:
- Questionnaires
- Collection of venous blood (50 ml)
- Collection of stool, urine, oral smear, and skin smear samples
- Two adipose tissue biopsies
- Ultrasonography of the carotid arteries
- Tonography of carotid and femoral artery
- Measurement ankle-brachial index
- ECG
- MRI of trunk and thigh
- MR spectroscopy of liver
- 30 minute automated blood pressure measurement
- 2 study visits (total 4 hours)
- Follow-up: annual telephone interview and questionnaire

Risks:
Hematoma related to collection of venous blood and adipose tissue biopsies.
Small scars (0.5 cm) of adipose tissue biopsies.