Microbiome in Healthy Perivascular Adipose Tissue

By

Murali Bollampally

A THESIS

Submitted to
Michigan State University
in partial fulfillment of the requirements
for the degree of

Basic Medical Science – Master of Science

2024

ABSTRACT

Perivascular adipose tissue (PVAT) is a novel

target

in the treatment of vascular

pathologies such as atherosclerosis and hypertension. It has been reported that the functions of

this

tissue include functions

in vasoprotective activity, anti-inflammatory roles, and

anti-contractile effects on the vessels that it surrounds. The lack of detailed investigation into the

form and function of this crucial tissue beckons further research into the composition of this

tissue. Comparisons between the structure of other adipose tissue throughout the body and PVAT

provide valuable information on the potential presence of bacteria, viruses or fungi within

healthy PVAT. This knowledge would enable an understanding of the changes that occur during a

disease state of the PVAT and further elucidate the function during the healthy state. Using this,

we propose further study into identifying a presence of microbiome within healthy PVAT tissue,

and argue that current evidence provides support for a presence of this microbiome.

TABLE OF CONTENTS

INTRODUCTION......................................................................................................................... 1

METHODS..................................................................................................................................... 2

RESULTS........................................................................................................................................3

DISCUSSION................................................................................................................................. 4

CONCLUSION.............................................................................................................................. 8

BIBLIOGRAPHY.......................................................................................................................... 9

iii

INTRODUCTION

The form and function of the perivascular adipose tissue (PVAT) is a burgeoning field of

study holding implications in vascular pathologies. Its composition can provide valuable clues

towards therapeutic modalities targeting these pathologies. Dysfunction of the PVAT and the

vasculature that it surrounds are key factors in diseases such as atherosclerosis and hypertension.

Kumar et al. identified a community of immune cells within the mesenteric and thoracic aorta

PVAT of Sprague Dawley rats (T cells, B cells, NK cells, and macrophages) (1). They also found

that these immune cell populations are similar in density to other non-PVAT fats, allowing for a

comparison between non-PVAT fats and PVAT fats to better understand the composition of

PVAT.

There is a large deficit in studies that investigate the presence of a microbiome (bacteria,

viruses or fungi) within any type of adipose tissue. Massier et al. is one of the few who showed a

presence of Proteobacteria and Firmicutes within human adipose tissue samples (2). This

presence of bacteria directly within adipose tissue additionally allows us to deliberate the

reasoning behind their existence, especially if there is a native microbiome present. Since

immune cells are present in PVAT, the additional presence of any type of microbiome within the

tissue would further elucidate the form and function of PVAT, which have not been fully studied.

If there is a presence of microbiome within the PVAT in a healthy state, we can probe deeper into

understanding why they are present and propose how this could change during states of vascular

dysfunction.

The interaction between commensal bacteria in the gut and the immune system is a useful

example that can be used to understand a potential presence of microbiome within PVAT. This

synergistic relationship whereby these bacteria and the immune system co-exist in a healthy state

is an explanation for finding bacteria in the PVAT.

Hypothesis: We hypothesize that there is a microbiome present in the PVAT, which is

crucial to the regular form and function of the PVAT.

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METHODS

This narrative review was written for publication within the Medical Hypotheses Journal

focusing on current research by searching PubMed and Google Scholar databases. Articles were

filtered based on presenting evidence of immune cells, bacteria, viruses or fungi within

perivascular adipose tissue or normal adipose tissue. Additional articles pertaining to the

composition and function of perivascular adipose tissue or methodologies to identify microbiota

within perivascular adipose tissue were included as well. As there is a relatively minute amount

of current research directly related to microbiome and perivascular adipose tissue, articles that

related to adipose tissue elsewhere in human or murine samples were included to provide support

to the comparison between adipose tissue and perivascular adipose tissue. The search used the

keywords: microbiome; perivascular adipose tissue; PVAT; adipose tissue; immune cells;

bacteria; viruses; fungi

After using the above methodology for the search, 38 relevant articles were found, of

which 17 were deemed relevant to support the hypothesis and future research on the subject.

Figure 1: Research article discovery strategy. 17 articles were found to build support and compel

future research to answer the questions: “Is there a presence of microbiome within healthy

perivascular adipose tissue?” and “How would dysfunction of this microbiome within

perivascular adipose tissue affect its normal form and function?”

2

RESULTS

Seventeen articles were identified as providing evidence that can be used to propose

support for a presence of microbiome within PVAT and answer the questions: “Is there a

presence of microbiome within healthy perivascular adipose tissue?” and “How would

dysfunction of this microbiome within perivascular adipose tissue affect its normal form and

function?”

Table 1: Methods used In Literature for Identifying Bacteria Within Adipose Tissue

Method

16S rRNA
Sequencing

Broad-range 16S
rRNA gene
hybrid capture

Tissue
Type/Location

Adipose Tissue
biopsy -
abdominal
subcutaneous,
omental-visceral
and
mesenteric-visce
ral

Staphylococcus
Aureus cultures

anti-Microbiota
IgG

Murine gut
culture and
blood samples

Main Finding

Limitations

Article

Lack of control
for translocation
of bacteria

Massier et al. (2)

Rassoulian
Barrett et al.
(17)

Time
consuming,
Need to control
for
contamination
within samples

Cross-reactivity Vujkovic-Cvijin

et al. (16)

Proteobacteria
and Firmicutes
were the main
phylum of
bacteria
discovered

Able to detect
bacterial strains
within a large
amount of
convoluting
human DNA

Translocating
bacteria from
mucosal surface
is able to be
detected by IgG

3

DISCUSSION

The PVAT is composed of varying types of adipose tissue. The aortic PVAT in mice is

similar to brown adipose tissue, whereas the mesenteric PVAT is similar to white adipose tissue

(3). This difference in type means that the composition may also vary depending on the location

of the PVAT being observed.

The PVAT is crucial

in guarding the vascular bed from inflammatory stress and

withstanding macrophage infiltration (3). Additionally, the PVAT is important in reducing the

stiffness of the aorta, which is valuable for conditions such as hypertension (4). Further

important for hypertension, the thoracic and mesenteric PVAT can also act as a reservoir for

norepinephrine, dopamine and serotonin (5). With these various functions, it is clear why

targeting the PVAT for therapeutic modalities is essential. If there is a microbiome, antibiotics

targeting the PVAT may be used in cases of acute inflammation, or in dysfunctions such as

hypertension or atherosclerosis. Diehm et al investigated the effect of the clindamycin and

cefazolin on adipose tissue graft samples taken from the upper thigh and abdomen. They showed

that

incubation of these antibiotics with the tissue samples led to decreased cell viability,

increased intracellular ROS production, and in high doses, cell rupture (6). However, they did

report that there was effective antibacterial activity in the samples. This raises the question of the

effect oral or intravenous antibiotic administration would have on adipose tissue composition and

would be an interesting point of investigation in PVAT if there is a microbiome within the PVAT.

Using antibiotics to target a dysfunctional microbiome in this tissue would provide a novel

method to combat disease in the vascular bed.

According to Kumar et al, there is a population of innate immune cells within murine

PVAT that was similar in density to murine non-PVAT fats. They identified six immune cell

types: B cells, Natural Killer cells, macrophages, mast cells, neutrophils and T cells (1). Zeng et

al. reported that regulatory T cells present in varying samples of human adipose tissue functioned

to regulate the immune and metabolic microenvironment in adipose tissue (7). Taking together

this evidence and the findings from Massier et al of a native bacterial population within human

abdominal subcutaneous, omental-visceral and mesenteric-visceral AT, we hypothesize that there

may be a subpopulation of bacterial microbiome within the PVAT that is co-existing with these

immune cells during a healthy state (2).

4

Only a few studies investigated the direct presence of microorganisms within adipose

tissue, and the most common limitation is the lack of investigation in healthy individuals.

Massier et al. studied adipose tissue samples from obese patients (2). The authors identified the

predominant presence of 16rRNA of Proteobacteria and Firmicutes phyla in abdominal

subcutaneous, omental-visceral, and mesenteric-visceral adipose tissue regions in patients

undergoing laparoscopic Roux-en-y bypass. However, the origin of these bacteria phyla was not

entirely clear. It has been speculated that there is a translocation of bacteria from the gut into

adipose tissue (2). A study utilizing healthy individuals taking into account the origin of the

bacteria would be beneficial to further understanding the presence of bacteria within PVAT and

normal adipose tissue.

He et al. found that

in mesenteric adipose tissue in patients with Crohn’s disease,

mesenteric adipose tissue resident Achromobacter pulmonis leads to the promotion of colitis (8).

The researchers found that Proteobacteria was the predominant phylum as found by 16s rRNA

sequencing, similar to the Massier et al. study (2). Although this was seen in a disease state, the

contribution of this microbiota towards furthering the disease progression could point towards an

exacerbation of pre-existing bacteria leading to these symptoms.

The complex interplay seen between commensal intestinal bacteria and immune cells

located within the intestinal epithelium provides a connection between the findings of immune

cells by Kumar et al. and findings of microbiota directly within adipose tissue from Massier et

al., allowing us to investigate the effect of this bacteria on the form and function of the PVAT.

The co-existence of bacteria and immune cells within the gut is a key presentation of how they

can co-exist in a healthy state.

Haller et al.

showed that when intestinal CaCO2 cells were challenged with

non-pathogenic E-coli and Lactobacillus sakei, there was a varying release of immune mediating

factors TNF-alpha and IL-beta compared to pathogenic bacteria (9). This finding points towards

a relationship whereby the aforementioned bacteria and immune cells were co-existing in a

non-disease state. Non-pathogenic commensal bacteria form a barrier of protection against

pathogenic bacteria and assist with proper digestion and nutrition absorption (10). Utilizing this,

a microbiome in PVAT could possibly provide resistance against pathogenic bacteria invasion

and support normal PVAT function.

5

The regular functions of the PVAT are mainly 1. Vasoprotective 2. Anti-inflammatory and 3.

Anti-contractile

For the vasoprotective and anti-inflammatory functions, there is a change during the

disease states of obesity and cardiovascular disease.The adipose tissue increases reactive oxygen

species and resistin, while decreasing release of adiponectin leading to vascular remodeling and

dysfunction (11). In a healthy state, the microbiome-immune system balance may be crucial in

maintaining a homeostasis and allowing for the regular release of these vaso-protective and

anti-inflammatory factors within PVAT. For the anti-contractile effects of PVAT that we see in

aortic vasculature, the PVAT is important in resisting contractility caused by TMAO which is

associated with increased risk for cardiovascular diseases (12). During the disease state of

obesity, this effect is reversed, with the PVAT releasing contractile factors seen in the thoracic

PVAT (13). Again, the balance between a native microbiome and the immune system may be

damaged, leading to the inflammation of the PVAT and release of these contractile factors.

With the majority of the evidence focused on adipose tissue and bacteria, it is also

valuable to consider other forms of microbiome (viruses and fungi) that may be present within

adipose tissue. Fungal-like particles in the form of lamarin-coated beads have been shown to

induce an inflammatory response within adipocytes (14). Adipose tissue acts as a reservoir for

viruses such as HIV (15). However, there is limited evidence connecting the presence of viruses

and fungi to PVAT or adipose tissue.

An important limitation within the literature to take into account during the investigation

of the hypothesis that there is a microbiome within the PVAT is that the bacteria is not native to

the PVAT, but rather has been translocated to the tissue from the gut microbiome. If this were the

case, the therapeutics may need to be targeted towards the gut rather than towards the PVAT.

Additionally, this translocation may alter the way we understand PVAT as the function may be

different if the bacteria were translocated during disease states rather than if it were native to the

PVAT in a healthy state. Massier et al. referenced a lack of biomarker availability in tracking the

origin of the discovered bacteria (2). However, Vujkovic-Cvijin et al. studied systemic IgG in

pro-inflammatory conditions where bacteria would be allowed to translocate across the gut

mucosal membrane and identified a plausible method of identifying pro-inflammatory bacteria

translocation from the gut (16). They presented that translocated murine commensal gut bacteria

lead to increased targeting by systemic IgG allowing them to essentially track the origin of the

6

bacteria from the gut (16). This could be a valuable method of understanding if there is a

translocation of gut bacteria in studies related to PVAT microbiome identification.

Another limitation evident in this study is the lack of evidence that directly connects a

presence of microbiome to perivascular adipose tissue. This is due to a general absence of

knowledge regarding PVAT as a whole. This however, highlights the importance of this paper as

it supports a further study into PVAT and its form and function.

7

CONCLUSION

A thorough investigation into all aspects of the form and function of PVAT is important

and necessary with vascular pathologies becoming ever more prevalent in today’s society.

Specifically, studies of these in healthy PVAT is important as minute changes in the delicate

homeostasis within the PVAT could lead to exacerbation of these disorders and may affect

current therapeutic modalities being used to combat these diseases. With this in mind, a presence

of microbiome in healthy PVAT is an important point of investigation that would be a valuable

piece to understanding this tissue and its effect on the vasculature further.

8

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