monoamine oxidase is a source of oxidative stress …draft 1 monoamine oxidase is a source of...

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MONOAMINE OXIDASE IS A SOURCE OF OXIDATIVE STRESS IN OBESE PATIENTS WITH CHRONIC INFLAMMATION

Journal: Canadian Journal of Physiology and Pharmacology

Manuscript ID cjpp-2019-0028.R1

Manuscript Type: Article

Date Submitted by the Author: 07-Apr-2019

Complete List of Authors: Sturza, Adrian; University of Medicine and Pharmacy Timisoara, Functional Sciences - Pathophysiology; University of Medicine and Pharmacy Timisoara, Center for Translational Research and Systems MedicineOlariu, Sorin; University of Medicine and Pharmacy Timisoara, Surgery II - 1st Clinic of SurgeryIonica, Mihaela; University of Medicine and Pharmacy Timisoara, Functional Sciences - PathophysiologyDuicu, Oana; University of Medicine and Pharmacy Timisoara, Functional Sciences - Pathophysiology; University of Medicine and Pharmacy Timisoara, Center for Translational Research and Systems MedicineVaduva, Adrian; University of Medicine and Pharmacy of Timisoara, Microscopic Morphology - MorphopathologyBoia, Eugen; University of Medicine and Pharmacy Timisoara, Pediatry - Pediatric SurgeryMuntean, Danina; University of Medicine and Pharmacy Timisoara, Functional Sciences - Pathophysiology; University of Medicine and Pharmacy Timisoara, Center for Translational Research and Systems MedicinePopoiu, Calin; University of Medicine and Pharmacy Timisoara, Pediatry - Pediatric Surgery

Is the invited manuscript for consideration in a Special

Issue:2018 IACS Slovakia

Keyword: monoamine oxidases, obesity, inflammation, endothelial dysfunction

https://mc06.manuscriptcentral.com/cjpp-pubs

Canadian Journal of Physiology and Pharmacology

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MONOAMINE OXIDASE IS A SOURCE OF OXIDATIVE STRESS IN OBESE

PATIENTS WITH CHRONIC INFLAMMATION

Adrian Sturza1,2,*, Sorin Olariu3,*, Mihaela Ionică1, Oana M. Duicu1,2, Adrian O. Văduva4,

Eugen Boia5, Danina M. Muntean1,2,#, Călin M. Popoiu5

1Department of Functional Sciences – Pathophysiology, 2Center for Translational Research

and Systems Medicine, 3Department of Surgery II - 1st Clinic of Surgery, 4Department of

Microscopic Morphology – Morphopathology, 5Department of Pediatry - Pediatric Surgery,

“Victor Babeș” University of Medicine and Pharmacy of Timișoara, Romania

#Corresponding author:

Danina M. Muntean, MD, PhD

Department of Functional Sciences - Pathophysiology,

Center for Translational Research and Systems Medicine,

“Victor Babeș” University of Medicine and Pharmacy of Timişoara, Romania

2, Eftimie Murgu Sq., 300041 Timisoara, RO

Tel: +40-256-493085

E-mail: [email protected]

*These authors contributed equally to this work.

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Abstract

Obesity is an important preventable risk factor for morbidity and mortality from

cardiometabolic disease. Oxidative stress (including in visceral adipose tissue) and chronic

low-grade inflammation are the major underlying pathomechanisms. Monoamine oxidase

(MAO) has recently emerged as an important source of cardiovascular oxidative stress. The

present study was purported to evaluate the role of MAO as contributor to reactive oxygen

species (ROS) production in white adipose tissue and vessels harvested from patients

undergoing elective abdominal surgery. To this aim, visceral adipose tissue and mesenteric

artery branches were isolated from obese patients with chronic inflammation and used for

organ bath, ROS production, qRT-PCR and immune-histology (IH) studies. The human

visceral adipose tissue and mesenteric artery branches contain mainly the MAO-A isoform, as

shown by the qRT-PCR and IH experiments. A significant up-regulation of MAO-A, the

impairment in vascular reactivity and increase in ROS production were found in obese vs

non-obese patients. Incubation of the adipose tissue samples and vascular rings with the

MAO-A inhibitor (clorgyline, 30 min) improved vascular reactivity and decreased ROS

generation. In conclusion, MAO-A is the predominat isoform in human abdominal adipose

and vascular tissues, is overexpressed in the setting of inflammation, and contribute to the

endothelial dysfunction.

Key words: monoamine oxidases, obesity, inflammation, endothelial dysfunction

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Introduction

Obesity is the most important modifiable risk factor for morbidity and mortality due to

life-threatening cardiovascular diseases, type 2 diabetes and certain cancers. With the

increased adoption of the sedentary and high-caloric intake lifestyles, particularly in

developing/low-income countries, the prevalence of overweight and obesity continues to rise

including among children (Fernandez-Sanchez et al. 2011; Hruby and Hu 2015). The

expanded adipose tissue is responsible for the inflammatory environment (Pereira and

Alvarez-Leite 2014) and high generation of reactive oxygen species (ROS), the two

pathomechanisms of obesity-related complications (McMurray et al. 2016). Indeed, the low-

grade chronic inflammation and oxidative stress at the level of adipose tissue (mainly

visceral) are the major contributors to the occurrence of type 2 diabetes mellitus (DM) and

accelerated atherosclerosis in the setting of obesity that currently affects 1/3 of people

worldwide, hence the term “globesity” (Afshin et al. 2017).

Several intracellular sites have been identified as being responsible for ROS

production, with the mitochondrial respiratory chain at the inner mitochondrial membrane

(Muntean et al. 2016; Murphy 2009) and NADPH oxidases being particularly involved

(Brandes and Schroder 2008).

In the past two decades, monoamine oxidases (MAOs) with 2 isoforms, A and B, at

the outer mitochondrial membrane have emerged as important sources responsible for

oxidative stress in the cardiovascular system in pathological conditions (Di Lisa et al. 2009;

Kaludercic et al. 2014b). These flavin-dehydrogenases catalyze the oxidative deamination of

neurotransmitters and biogenic amines (serotonin, dopamine and norepinephrine) with the

constant generation of hydrogen peroxide, aldehydes and ammonia as deleterious by-products

(Di Lisa et al. 2009; Kaludercic et al. 2014a).

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MAO-A is classically considered the major isoform in the cardiovascular system that

increases with ageing and is responsible for the related cardiac oxidative damage (Kaludercic

et al. 2011) and also mediates the maladaptive evolution of ventricular hypertrophy towards

heart failure (Kaludercic et al. 2014a), the endothelial dysfunction in the setting of

hypertension (Sturza et al. 2013a; Sturza et al. 2013b), inflammation (Sturza et al. 2013a),

chronic kidney disease (Utu et al. 2017) and diabetes (Duicu et al. 2016; Lighezan et al. 2016;

Sturza et al. 2015b).

Scarce literature is available on MAOs contribution to oxidative stress in the adipose

tissue. There are a couple of studies that reported an increased MAO activity in the white

adipose tissue in obese mice (Tong et al. 1979) and dogs (Wanecq et al. 2006). As for

humans, a pioneering study from the Parini's group identified high monoamine oxidase

activities in abdominal and mammary human adipocytes and reported their role in the

clearance of plasma norepinephrine (Pizzinat et al. 1999). More recently, MAO expression

was reported to be increased during adipogenesis in human adipocytes (Bour et al. 2007).

However, no data are available concerning the role of these enzymes in the visceral

adipose tissue in obesity associated with systemic inflammation. Thus, the aim of the present

study was to investigate the MAO contribution to oxidative stress in adipose tissue and

vascular samples harvested from patients with obesity and chronic inflammation.

Material and methods

Samples of omental adipose tissue and mesenteric arteries were obtained from

consecutive patients undergoing abdominal surgery. Patients were randomized into two

groups: (1) obese patients (OB) with an inflammatory status (n=10; 4 women, 6 men), and

(2) non-obese (nonOB) without inflammation (n=10; 5 women, 5 men), respectively.

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The University Committee for Research Ethics approved the study protocol (nr.

11/31.03.2017) and the informed consent was obtained from all patients prior to surgery,

according to the World Medical Association Declaration of Helsinki.

Immune-Histochemistry Studies

Tissue expression of the MAO was quantified in frozen sections of human intra-

abdominal adipose tissue and branches of mesenteric arteries using the MAO-A (Abcam,

ab126751, 1:50) primary antibody and Alexa Fluor labeled secondary goat anti-rabbit

antibody (Invitrogen, A32731, 1:200), respectively, as previously described (Sturza et al.

2015a; Sturza et al. 2015b; Sturza et al. 2018). Nuclear staining was obtained with DAPI

(Santa Cruz, SC3598). The slides were examined using an Olympus Fluoview FV1000

confocal microscope (DAPI ex/em 405/461nm, Alexa Fluor ex/em 543/612nm). Images were

analyzed with Icy, a free open source image analysis software, developed by the Quantitative

Image Analysis Unit at Institute Pasteur (Paris) (de Chaumont et al. 2012).

Oxidative Stress Assessment By Means of Ferrous Iron Xylenol Orange Oxidation Assay

Hydrogen peroxide production was assessed in samples of visceral adipose tissue and

branches of mesenteric arteries in the presence vs. absence of the MAO-A inhibitor,

clorgyline (10 μM, 30 min incubation) using the Ferrous iron xylenol orange OXidation

(FOX) assay (PeroxiDetect Kit, Sigma Aldrich) as previously described (Danila et al. 2017;

Lighezan et al. 2016; Sturza et al. 2015b; Sturza et al. 2013a; Sturza et al. 2015c; Sturza et al.

2018; Utu et al. 2017). The principle of the assay is that peroxides oxidize Fe2+ to Fe3+ ions at

acidic pH. The Fe3+ ion will form a colored adduct with xylenol orange, which is

spectrophotometrically measured at 560 nm. Subsequently the production of H2O2 was

calculated using the standard curve. The result is expressed in nmol H2O2/h/mg tissue.

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Oxidative Stress Assessment By Means of Immune-Fluorescence

The total amount of ROS was determined using the DHE (dihydroethidium) probe

according to a previously described technique (Duicu et al. 2016; Miller et al. 2008). Briefly,

the samples were embedded in OCT and snap-frozen. The frozen fragments were cut in 8 μm

thick cryosections and put on glass slides. After 3 washes with PBS, 5 minutes each, the

cryosections were incubated in the dark with DHE for 30 minutes at room temperature.

Excess DHE was removed by 3 additional washes with PBS. The slides were mounted with

Vectashield (Vector Laboratories) and immediately analyzed in confocal microscope

(Olympus Fluoview FV1000). Images were obtained using laser excitation at 488 nm. Image

analysis was performed using Icy Bioimage Analysis software (de Chaumont et al. 2012).

Real Time Polymerase Chain Reaction (RT-PCR)

All tissues were homogenized using the Tissue Lyser (Qiagen).Total RNA was

isolated (Total RNA Mini SI Isolation Spin-Kit, Applichem), measured using was verified

using a Nanodrop 2000 spectrophotometer (Thermo Scientific) and used for reverse

transcription (Superscript III RT, Invitrogen). Quantitative RT-PCR (Biorad) was performed

in adipose tissue and vascular samples. Primers against MAO isoforms were designed using

sequence information from the NCBI database (5’→ 3’): human MAO-A forward: 5′-CTG

ATC GAC TTG CTA AGC TAC-3′, human MAO-A reverse: 5′-ATG CAC TGG ATG TAA

AGC TTC-3′. The housekeeping gene (EEF2, eukaryotic elongation factor 2) and its primers

were as follow (5’→ 3’): EEF2 forward: GAC ATC ACC AAG GGT GTG CAG and EEF2

reverse: GCG GTC AGC ACA CTG GCA TA.

Vascular Reactivity Studies

The vascular samples (mesenteric artery branches) were mounted in the myograph

(DMT) chambers filled 5 ml of Krebs solution (37°C) aerated with 95% O2-5% CO2 gas

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mixture (pH 7.4). The rings were stretched to an optimal tension of 1 g, allowed to equilibrate

for 45 min, and further exposed to 80 mM KCl. The concentration of phenylephrine used for

preconstriction was adjusted to obtain a preconstriction level of 80% of the contraction

elicited by KCl (80 mM). Endothelium-dependent relaxation to cumulative concentrations of

acetylcholine (Ach) and contractility to nitric oxide synthase (NOS) inhibitor L-NAME

(10μM) were recorded in the presence vs. absence of irreversible MAO-A inhibitor, clorgyline

(10 µM). Nitric oxide (NO) bioavailability was estimated from the constrictor response (%) to

the classic NO synthases inhibitor, N-nitro-L-arginine (L-NAME, 10 µM) in vascular rings

preconstricted with phenylephrine to 10% of the maximal KCl constriction.

Statistics

Data are presented as mean±SEM and were analyzed using a one-way ANOVA or

student t-test when appropriate. Data analysis of the dose-effect response curves was

performed using the ANOVA F-test (comparisons of bottom and top values, EC50 and the

Hill slope). Values of p

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Limited information is available regarding the MAO expression in human visceral

adipose tissue and mesenteric vessels. For this reason we evaluated the expression of MAO

isoforms in adipose tissue samples and arterial fragments isolated from patients subjected to

elective abdominal surgery by means of IH. The results showed that both MAO isoforms were

present with predominance of the MAO-A (Fig. 1A). MAO was diffusely distributed in the

adipose tissue whereas in the vascular wall we noticed an increased expression in the intima

and the adventitia (Fig. 1A).

Obesity And Chronic Inflammation Are Associated With Increased MAO-A Expression In

Both Adipose Tissue And Vasculature

Increased oxidative stress in the adipose tissue and vasculature is the hallmark of

overweight and obese patients but the sources of ROS are partially elucidated. While the

classical sources still remain NADPH oxidases and the mitochondrial respiratory chain, we

report here that MAO-A isoform contributes to the adipose tissue and vascular oxidative

stress, being up-regulated in samples isolated from obese patients with chronic inflammation,

as revealed by the mRNA gene expression (Fig. 1B).

MAO-A Is A Source Of ROS in Adipose Tissue And Vasculature When Obesity Coexists With

Inflammation

As MAO-A was up-regulated in the adipose tissue and vascular preparations from

obese patients we further assessed the ROS production by IH and FOX assay after incubating

the samples with the MAO-A inhibitor, clorgyline (10 μM, 30 min). Ex vivo incubation with

clorgyline was able to reduce the signal related to oxidative stress as revealed by DHE

staining (Fig. 2A) and rate of hydrogen peroxide production measured by FOX assay (Fig.

2B).

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MAO Inhibition Improves Vascular Reactivity of Mesenteric Branches Isolated From Obese

Patients With Inflammation

Since we noticed an upregulation of MAO-A together with an increased amount of

ROS in the vessels we thought to determine whether MAO inhibition is also able to modulate

the reactivity of the vascular preparations in organ bath studies. Vascular contractility in

samples obtained from obese patients with inflammation was significantly increased in

response to cumulative doses of phenylephrine whereas the endothelium-dependent relaxation

was significantly attenuated. Incubation with the irreversible MAO-A inhibitor restored both

the contractile and relaxation responses. Also, contractility to L-NAME (Nω-Nitro-L-arginine

methyl ester hydrochloride, 10 μM), the classic NO synthases inhibitor, was significantly

increased in samples from obese patients; this response was reduced after clorgyline, a finding

that strongly suggests the involvement of NO in the vascular protective effect of MAO

inhibition.

Discussions

The main finding of this pilot study is that MAO-A isoform contributes to the adipose

tissue and vascular oxidative stress in the setting of obesity and systemic inflammation.

These observations are particularly important since a persistent, dysfunctional low-grade

inflammatory status is the hallmark not only of obesity but also of all non-communicable

chronic diseases of the 21st century (Bennett et al. 2018). The obesity-driven changes of the

adipose tissue microenvironment are responsible for the chronic systemic inflammation via

the upregulation of the pro-inflammatory cytokines and the downregulation of the anti-

inflammatory ones, as recently reviewed in (Fuster et al. 2016).

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Also, the increased oxidative stress in the setting of obesity has been systematically

investigated, in particular the role dysfunctional complexes of the electron transport chain

(McMurray et al. 2016). Here we unequivocally demonstrated that MAO is an important

contributor to the oxidative stress in obese patients with chronic inflammation. Indeed, MAO

inhibition was able to significantly reduce the amount of H2O2 in the white adipose tissue and

vascular samples from obese patients. Of note, in a previous study we firstly noticed the

presence of MAO in the perivascular adipose tissue of coronary arteries isolated from patients

with coronary artery disease (Lighezan et al. 2016) - an observation that prompted this study.

In our hands, ex vivo MAO inhibition elicited beneficial effects in several studies performed

in human vascular samples, such as internal mammary arteries isolated from patients

subjected to coronary by-pass surgery (Lighezan et al. 2016) and in collaterals of brachial

artery in patients with chronic kidney disease with indication of hemodialysis (Utu et al.

2017).

Interestingly, irreversible MAO-A inhibition with clorgyline elicited a significant

reduction of oxidative stress only in adipose tissue samples harvested from the obese group as

revealed by the DHE staining and FOX assay, respectively. However, in organ bath

experiments, clorgyline was able to reduce contractility in all the vascular rings, regardless the

study group. This observation is suggestive for both tissue specificity and pleiotropic effects

of MAO inhibitors (behind MAO inhibition). In an earlier study we investigated whether

MAO inhibitors act as ROS scavengers, a hypothesis that was not confirmed (Sturza et al.

2013a). However, since clorgyline was able to reduce the contractility to L-NAME, we

speculate that its beneficial effect is mediated via an interaction with the NO-dependent-

pathway. In this respect, we have previously observed in HUVECs that MAO directly

interfere with NO signaling pathway and limit the accumulation of cGMP (Sturza et al.

2013a).

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The presence of MAO-A has been documented together with the one of semicarbazide

sensitive amine oxidase (SSAO) in the mesenteric perivascular adipose tissue from rats

(Ayala-Lopez et al. 2017). Interestingly, these authors reported that individual inhibition of

either enzyme did not alter the contractility to norepineprine; however, inhibition of both

MAO and SSAO increased the effect of norepinephrine on mesenteric arteries with peripheral

adipose tissue. We acknowledge as limitations of our study the fact that we did not assess the

enzymatic activity and protein expression or putative crosstalk with other amine oxidases.

Also, the level of free fatty acids which may directly interfere (when increased) with the

vascular reactivity via a direct membrane effect was not measured.

The constant interest in studying ROS sources in the cardiovascular system stems from

the fact that cardiovascular diseases and their comorbidities (obesity, metabolic syndrome and

diabetes mellitus) remain the leading causes of morbidity and mortality worldwide, despite a

huge and constantly expanding therapeutic armamentarium. All of these pathologies have

common pathogenesis that include the triple association of endothelial dysfunction, "low-

grade" inflammation and oxidative stress, all cumulatively contributing to disease progression

and complications. Nevertheless, whether the increased MAO expression in the visceral

adipose tissue is a cause or a consequence of the prolonged low-grade inflammatory status

remains an open question.

MAO inhibitors have been systematically studied starting from the mid-past century

for their therapeutic efficacy in neurodegenerative and psychiatric illnesses (including

depression) via complex mechanisms that include the mitigation of the oxidative stress

(Bortolato et al. 2008; Song et al. 2013). Therefore, in line with the recently reported

association between a higher body mass index (BMI) and depression, particularly in women

(Tyrrell et al. 2018) the study of the new generations of reversible MAO inhibitors as

potential candidates for drug repurposing in the treatment of cardiometabolic diseases

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represents a feasible alternative. A starting point could be represented by the assessment of

the effects of MAO inhibition on the systemic inflammatory status in the setting of chronic

non-communicable diseases.

Conclusions

In conclusion, monoamine oxidases are expressed in human visceral adipose tissue

and branches of mesenteric artery and the MAO-A isoform is upregulated in the setting of

obesity associated with chronic inflammation. In vitro inhibition of MAO-A significantly

reduced the amount of oxidative stress in both adipose tissue and arteries; in the latter an

improved vascular reactivity (reduced contractility and increased endothelium-dependent

relaxation) was also found. These data also suggest that MAO inhibitors are promising

candidate compounds for drug repositioning.

Acknowledgements

Research supported by the university grant PIII-C5-PCFI-2017/2018-01.

Conflicts of interest

The authors declare that there is no conflict of interest regarding the publication of this paper.

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Figure captions

Figure 1. MAO is expressed in human adipose tissue and vasculature and is up-

regulated in setting of obesity. (A) MAO expression in human intra-abdominal adipose

tissue and mesenteric artery branches in Immune-fluorescence: Green - anti-MAO-A

antibody, blue - DAPI. (B) MAO-A expression (fold change) in visceral adipose tissue and

mesenteric artery branches from obese (OB) and non-obese (nonOB) patients – qPCR,

*p

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Table 1. Characteristics of the study groups.

OB

(BMI≥30 kg/m2)

n=10

(4 ♀, 6♂)

non-OB

(BMI

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Hb A1c (%) 5.2±0.3 5.3±0.14 Ns

ALAT (U/l) 32.4±6.2 28±2 Ns

ASAT(U/l) 22.6±5.56 20±3 Ns

Total serum calcium (mg/dl) 8.6±0.45 8.72±0.4 Ns

Ionized calcium (mg/dl) 4±0.34 4.89±0.5 Ns

Serum phosphate (mg/dl) 5.2±0.3 5.3±0.14 Ns

Data are means ± S.E.M. and differences are highlighted in bold.

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Figure 1. MAO is expressed in human adipose tissue and vasculature and is up-regulated in setting of obesity. (A) MAO expression in human intra-abdominal adipose tissue and mesenteric artery branches in Immune-fluorescence: Green - anti-MAO-A antibody, blue - DAPI. (B) MAO-A expression (fold change) in

visceral adipose tissue and mesenteric artery branches from obese (OB) and non-obese (nonOB) patients – qPCR, *p

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Figure 2. MAO is a source of oxidative stress is human adipose tissue and vasculature. The effect of MAO-A inhibitor clorgyline (Clorg, 10 μM) on oxidative stress in (A) adipose tissue and (B) mesenteric artery

branches samples harvested from obese (OB) and non-obese (nonOB) patients – DHE stain. (C) The effect of MAO-A inhibitor clorgyline (Clorg, 10 μM) on hydrogen peroxide production in adipose tissue and

mesenteric artery branches from obese (OB) and non-obese (nonOB) patients – FOX assay. *p

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Figure 3. MAO-A inhibition improves vascular reactivity in human mesenteric artery branches.(A) Phenylephrine-induced contractions, (B) Acetylcholine-induced endothelium-dependent relaxation (C) Contraction to L-NAME (Nω-Nitro-L-arginine methyl ester hydrochloride, 10μM), in human mesenteric

arteries branches obtained from obese (OB) and non-obese (nonOB) patients, incubated or not with MAO-A inhibitor, clorgyline (Clorg, 10 μM, 30 min). *p

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