Endothelial function and gene expression in perivascular adipose tissue from internal mammary arteries of obese patients with coronary artery disease

Accepted Manuscript Endothelial function and gene expression in perivascular adipose tissue from internal mammary arteries of obese patients with coronary artery disease Maria Cybularz, Heike Langbein, Birgit Zatschler, Coy Brunssen, Andreas Deussen, Klaus Matschke, Henning Morawietz PII:

S1567-5688(17)30084-3

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10.1016/j.atherosclerosissup.2017.05.042

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Please cite this article as: Cybularz M, Langbein H, Zatschler B, Brunssen C, Deussen A, Matschke K, Morawietz H, Endothelial function and gene expression in perivascular adipose tissue from internal mammary arteries of obese patients with coronary artery disease, Atherosclerosis (Supplements) (Component) (2017), doi: 10.1016/j.atherosclerosissup.2017.05.042. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

ACCEPTED MANUSCRIPT Title: Endothelial function and gene expression in perivascular adipose tissue from internal mammary arteries of obese patients with coronary artery disease

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Running head: Endothelial function in obese patients

Author names and affiliations:

Maria Cybularz1, Heike Langbein1, Birgit Zatschler2, Coy Brunssen1, Andreas Deussen2,

Division of Vascular Endothelium and Microcirculation, Department of Medicine III,

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Klaus Matschke3, Henning Morawietz1

University Hospital Carl Gustav Carus, Technische Universität Dresden, Fetscherstr. 74, 01307 Dresden, Germany 2

Institute of Physiology, Medical Faculty Carl Gustav Carus, Technische Universität Dresden,

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Fetscherstr. 74, 01307 Dresden, Germany

Department of Cardiac Surgery, Herzzentrum Dresden, Medical Faculty Carl Gustav Carus,

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Technische Universität Dresden, Fetscherstr. 76, 01307 Dresden, Germany

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Corresponding author:

Prof. Dr. Henning Morawietz, Division of Vascular Endothelium and Microcirculation, Department of Medicine III, University Hospital and Medical Faculty Carl Gustav Carus, TU Dresden, Fetscherstr. 74, 01307 Dresden, Germany Tel.: +49-351-458-6625, FAX: +49-351-458-6354, E-Mail: [email protected]

Word count of body: 4.765

Tables: 2

Figures: 3

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ACCEPTED MANUSCRIPT Abstract

Background and Aims: Obesity is a risk factor for endothelial dysfunction and atherosclerosis. However, perivascular adipose tissue can release adipokines and other unknown adiposederived relaxing factors. Therefore, we investigated the impact of obesity on vascular function

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and expression of genes in perivascular adipose tissue from internal mammary arteries of patients with coronary artery disease undergoing coronary artery bypass grafting.

Methods: The vessel function was compared between groups of patients with a body-mass

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index (BMI) between 25-30 kg/m2. The groups did not differ in age, gender (males), and ejection fraction. Vascular segments of internal mammary arteries were examined in a

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Mulvany myograph. Following preconstriction with noradrenaline, dose-response curves were assessed for relaxation with acetylcholine and sodium nitroprusside. Results: Maximum contraction in response to potassium and noradrenaline was increased in obese patients with a BMI > 30 kg/m2. EC50 of endothelium-dependent relaxation was

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impaired in patients with a BMI above 25, but below 30 kg/m2. Sodium nitroprussidemediated maximal relaxation was not different between study groups. Integrin alpha X chain (ITGAX/CD11c) and macrophage mannose receptor (MRC1/CD206) expression was reduced

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in perivascular adipose tissue of patients with a BMI above 30 kg/m2, while adiponectin (ADPQ) expression was increased in the same tissue.

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Conclusion: Our data suggest a partially reduced endothelial function in internal mammary arteries of adipose patients with a BMI between 25 and 30 kg/m2 undergoing coronary artery bypass grafting surgery. Increased adiponectin expression in perivascular tissue might contribute to maintenance of endothelial function in obese patients with a BMI above 30 kg/m2. Supplementary key words endothelial function, perivascular adipose tissue, obesity, coronary artery disease

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ACCEPTED MANUSCRIPT Introduction

The prevalence of obesity is globally increasing and associated with a higher mortality [1, 2]. Obesity is a major risk factor of cardiovascular diseases [3]. It can elevate the prevalence of coronary artery disease and the atherothrombotic risk of patients [4]. Obesity-driven adipose

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tissue dysfunction can promote a chronic inflammatory state [5]. This is characterized by abnormal adipokine production and activation of proinflammatory pathways [6]. Another possible pathomechanism is the increased infiltration of macrophages and lymphocytes into

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the adipose tissue resulting in activation of chronic inflammation and of transcription factors orchestrating the metabolic responses to obesity [6]. This abnormal accumulation of inflamed

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adipose tissue can have deleterious effects on vascular tone and remodeling [7]. However, increasing evidence supports a vasorelaxing and vasoprotective role of perivascular adipose tissue [7, 8]. Through production of adipokines and the release of other thus far unidentified factors, adipose tissue modulates vascular tone regulation [9]. Despite the

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potential adverse impact of overweight and obesity, recent epidemiological data have demonstrated an association of mild obesity and, particularly, overweight on improved survival [10]. This might contribute to the obesity paradox [10, 11] and leads to new concepts

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like the fat-but-fit paradigm and the metabolically healthy but obese phenotype in patients with cardiovascular diseases [12]. However, the association of the body-mass index with

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vascular structure and function of internal mammary arteries and its adipose perivascular tissue is not well-understood. Therefore, we investigated the impact of obesity on vascular function and expression of genes in perivascular adipose tissue from internal mammary arteries of patients with coronary artery disease undergoing coronary artery bypass grafting.

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ACCEPTED MANUSCRIPT 2. Methods 2.1. Patients

The vascular function was compared between groups of patients with a body-mass index (BMI) below 25 (lean, n=18), between 25 and 30 (adipose, n=25) and above 30 kg/m2 (obese,

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n=24). Selection criteria of the patients were coronary artery disease with the indication for coronary artery bypass grafting (CABG) surgery and male gender. Patients underwent elective CABG surgery and distal remnant specimens of the left internal mammary artery

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obtained after informed consent from patients were analyzed. In these patients, endothelial function was determined in internal mammary artery rings by organ chamber experiments. In

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further studies, gene expression in the perivascular adipose tissue was analyzed. All participants received detailed verbal and written information about the study objectives and procedure, and gave written informed consent. The study was approved by the Ethics Committee of the Technische Universität Dresden (EK 307122007). The investigation

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conforms with the principles outlined in the Declaration of Helsinki (1997).

2.2. Isolation of segments of internal mammary arteries

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We isolated left human internal mammary arteries (A. thoracica interna) from male patients undergoing coronary artery bypass grafting surgery in the Heart Center Dresden. After

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median sternotomy, the artery was carefully dissected and a distal segment was donated for our studies. In order to maintain a better endothelial and smooth muscle function, all vessels were stored after surgery in a recently developed potassium-chloride and N-acetyl-histidineenriched storage solution at 4°C (TiProtec®, Dr. F. Köhler Chemie, Bensheim, Germany) and immediately transported to the laboratory within 10 minutes. While separating from connective tissue and perivascular adipose tissue under a microscope (Fig. 1A, B), the vessels were kept in the protective solution in a silicon pale. The arterial vessels were divided into 3 parts. The vessel function of the middle segment was measured in

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a Mulvany myograph. The second segment was frozen in liquid nitrogen for further mRNA isolation and real-time PCR. The third segment was stored in formalin for histological staining. The separated perivascular tissue was also immediately frozen in liquid nitrogen for

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future mRNA isolation and real-time PCR.

2.3. Assessment of vascular function

Vascular segments of left human internal mammary arteries were examined as previously

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described [13] by modification of protocols for experimental models [2, 14] and human vessels [15] in a Mulvany myograph (Modell 610M, Danish Myo Technology, Aarhus,

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Denmark). Following preconstriction with noradrenaline, dose-response curves were assessed for relaxation with acetylcholine. Endothelium-independent maximal smooth muscle cell relaxation was determined with sodium nitroprusside.

In brief, following a 15-minute period of stabilization, the solution was removed from the

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organ bath and replaced by high potassium physiological salt solution in order to depolarize the smooth muscle cells membrane and provoke a maximal receptor-independent contraction. Next, the organ bath and the vascular rings were washed four times with physiological sodium

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chloride solution. Following preconstriction with 10 µM noradrenaline, dose-response curves were assessed for relaxation with acetylcholine and sodium nitroprusside. All measurements

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were performed by a colleague initially blinded to patient group assignment.

2.4. RNA isolation and real-time PCR RNA was isolated from frozen tissue using the peqGOLD Total RNA Kit (PEQLAB Biotechnologie GmbH, Erlangen, Germany). The RNA concentration was determined using the NanoDrop™ 1000 Spectrophotometer (PEQLAB Biotechnologie GmbH, Erlangen, Germany). RNA was transcribed by reverse transcription into cDNA. In brief, 500 µg RNA, 1 µl of random primer, 1 µl of dNTPs and DEPC-treated water (final volume 12.5 µl) were

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mixed for each sample. All samples were incubated in an Eppendorf Mastercycler Personal 5332 for 5 minutes at a temperature of 65ºC. Next, 7.5 µl of MasterMix, containing 5xBuffer, DDT, RNAse OUT and Superscript reverse transcriptase (Invitrogen, Karlsruhe, Germany) were added to each sample. The samples were incubated at 25ºC for 10 minutes, at 42ºC for

DEPC-treated water to a cDNA concentration of 10 ng/µl.

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50 minutes and finally at 70ºC for 15 minutes, transferred to ice and diluted with 30 µl of

Real-time PCR was performed using the 7500 Fast Real-Time PCR System (Applied

(ITGAX/CD11c,

macrophage

type

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marker),

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Biosystems, Foster City, CA, USA). The mRNA expression of integrin alpha X chain macrophage

mannose

receptor

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(MRC1/CD206, macrophage type 2 marker), adiponectin (ADPQ) and leptin (LEP) was measured in RNA from perivascular adipose tissue obtained from internal mammary arteries by real-time polymerase chain reaction (PCR) as described [13, 16-18]. In addition, we determined the expression of alpha smooth muscle actin (alpha-actin-2, ACTA2) as marker of

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vascular smooth muscle cells in internal mammary arteries. PCR primers are summarized in Tab. 2. Analysis of raw data was performed with 7500 Software version 2.06 (Applied Biosystems by Life Technologies, Darmstadt, Germany). Evaluation of the data was done

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using a mathematical model of relative expression ratio in real-time PCR under constant

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reference gene expression [19].

2.5. Analysis of vascular histology Internal mammary artery segments were fixed in phosphate-buffered paraformaldehyde (4 % PFA), dehydrated and embedded in paraffin. Serial 5 µm sections were cut, mounted on glass slides, deparaffinized and rehydrated through degraded ethanol. Vessel sections were stained with Elastica van Gieson staining technique as described [17]. Entellan was used as a mounting medium. Sections were photographed with Zeiss Axio Observer.Z1 ApoTome

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microscope. In each section, the following parameters were measured using the Fiji imaging software: total area, area of the media, area of the intima, intima/media ratio.

2.6. Statistics

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Data are shown as means ± SD. Statistical analysis was performed by One-Way ANOVA followed by Bonferroni’s method (Sigma Stat 3.11, Systat Software, Inc., San Jose, CA,

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USA). A value of P<0.05 was considered statistically significant.

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ACCEPTED MANUSCRIPT 3. Results 3.1. Analysis of clinical parameters

The groups of patients with a body-mass index (BMI) below 25 (lean), between 25 and 30

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(adipose) and above 30 kg/m2 (obese) analysed for vascular function did not differ in age, gender (males), ejection fraction (EF), smoking status, prevalence of hypertension and dyslipidemia and additional anti-hypertensive and statin therapy (Tab. 1). The group of obese

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patients had a higher prevalence for type 2 diabetes mellitus. The groups of patients analysed for gene expression in perivascular adipose tissue did not differ in medical therapy and

3.2. Assessment of vascular function

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clinical parameters except BMI (data not shown).

The vessel wall tension after contraction with potassium chloride and noradrenalin was significantly increased in adipose patients with BMI above 30 kg/m2 compared to groups of

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lean and obese patients (Fig. 1C, D). Endothelium-dependent vascular relaxation showed a right-shift (Fig. 1E). The EC50 of endothelium-dependent vascular relaxation was impaired in

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the adipose group (BMI between 25-30 kg/m2) (Fig. 1F). We found no significant differences in sodium-nitroprusside-mediated maximal relaxation between the study groups with different

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BMI (Fig. 1G).

3.3. Vessel histology

Representative images of sections of the internal mammary arteries of patients with different BMI are shown in Fig. 2A-C. We found no significant differences in relative media area and intima/media ratio between the study groups of patients with different BMI (Fig. 2D, E). This was further supported by analysis of expression of the marker of vascular smooth muscle cells alpha-actin 2 (ACTA2), which did not differ between all 3 study groups (Fig. 2F).

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ACCEPTED MANUSCRIPT 3.4. Gene expression in perivascular adipose tissue

In order to get further insight in the putative impact of perivascular adipose tissue on vascular function, we analyzed the gene expression of different marker genes and adipokines. Integrin alpha X chain (ITGAX/CD11c) as a marker of M1 macrophages was significantly reduced in

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perivascular adipose tissue of obese patients with coronary artery disease and a BMI above 30 kg/m2 (Fig. 3A). Surprisingly, the expression of the M2 marker macrophage mannose receptor 1 (MRC1/CD206) was significantly reduced in the perivascular adipose tissue of obese

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patients as well (Fig. 3B).

Adipokine adiponectin (ADPQ) was significantly induced in the same amount of perivascular

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adipose tissue of obese patients with a BMI above 30 kg/m2 compared to lean (BMI <25 kg/m2) and adipose patients (BMI between 25 and 30 kg/m2) (Fig. 3C). It is noted, however, that this elevation of adiponectin expression was quite heterogeneous in obese patients. In contrast, leptin (LEP) expression in perivascular adipose tissue was not significantly different

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between the patient groups with different BMI (Fig. 3D).

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ACCEPTED MANUSCRIPT Discussion

In this study, we analyzed the impact of body-mass index (BMI) on vascular function and gene expression in the perivascular adipose tissue of internal mammary arteries of patients undergoing coronary artery bypass grafting (CABG) surgery. The study groups did not differ

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in most clinical parameters and medical therapy. Nevertheless, other non-determined clinical parameters might have an impact on the vascular function in the individual patients as well. The prevalence of diabetes mellitus type 2 was higher in the group of obese patients with a

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BMI > 30 kg/m2 analyzed for vascular function. Therefore, the differences observed between groups of adipose and obese patients may also have been due to an impaired glucose handling

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in obese patients. Obesity is a well-established risk factor of type 2 diabetes and coronary artery disease [12]. Therefore, the even slightly better preserved endothelium-dependent vascular function compared to adipose patients (BMI 25-30 kg/m2) was unexpected (Figure 1E, F). Because vessel segments from adipose patients reacted with a stronger constriction in

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response to potassium and noradrenalin, this different level of constriction may have affected the consecutive measurement of endothelium-dependent relaxation. However, vasorelaxation induced by sodium nitroprusside was similar between both groups (Figure 1G), making this

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explanation unlikely. On the other hand, several experimental and clinical data support an impaired endothelial function in diabetes. Therefore, a major impact of the higher prevalence

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for type 2 diabetes on the observed even slightly better vascular function compared to adipose patients (BMI 25-30 kg/m2) is unlikely. The stronger contraction in response to potassium chloride and noradrenaline in obese patients with a BMI above 30 kg/m2 compared to lean patients supports a higher vessel wall tension in response to obesity. Adipose patients with a BMI of 25-30 kg/m2 did not show significantly increased vessel wall tension. Endothelium-dependent relaxation was impaired in the adipose group with a BMI between 25 and 30 kg/m2. This supports an endothelial dysfunction in adipose patients compared to lean and obese patients.

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We can only speculate about the impact of the novel TiProtect solution in this context. This solution leads to a well-preserved endothelial function [15]. This might partially explain differences to previous studies which showed stronger impairment of vascular function in

a better representation of the endothelial function in vivo.

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obesity [5, 12]. However, a vessel function well-preserved during cold storage should result in

The observed changes could be due to structural changes in the vessel wall. Therefore, we examined the histology of tissue sections of internal mammary arteries. No significant

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differences were observed in relative media area and intima/media ratio between the study groups. In addition, expression of the marker of vascular smooth muscle cells alpha actin 2

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was not changed. This argues against different degrees of vascular hypertrophy in the studied vessels. It is unclear to which extent this reflects vessel wall composition in other arterial beds, because the internal mammary artery is known to exhibit relatively little structural remodeling in patients undergoing bypass surgery. We did not observe major atherosclerotic

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changes in the vascular sections. This is in agreement with the lower susceptibility of the internal mammary arteries to atherosclerosis in comparison to coronary arteries. The perivascular adipose tissue is an interesting novel regulator of vascular function. Obesity

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may lead to activation of adaptive vascular mechanisms to enhance dilatory function of different arterial vessels in hypertensive patients [20]. Several potential mechanisms are

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currently discussed [8, 9]. They include NO availability, oxidative stress due to increased infiltration with inflammatory macrophages as major sources of reactive oxygen species, physiological concentrations of endothelium-derived vasorelaxing factors like hydrogen peroxide and hydrogen sulfide or the release of so far unknown adipose-tissue derived relaxing factors. Due to limited availability of human tissue we focused in this study on the expression of marker genes in the perivascular adipose tissue. First, we tested whether an inflamed perivascular adipose tissue with increased infiltration by M1 macrophages is a major source

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of endothelial dysfunction. We found a reduced expression of the M1 marker integrin alpha X chain (ITGAX/CD11c) in perivascular adipose tissue of obese patients with a BMI > 30 kg/m2. This might at least partially explain the surprisingly well-preserved vascular function in this group of patients. In parallel, we found a reduced expression of M2 marker

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macrophage mannose receptor 1 (MRC1/CD206) in the perivascular adipose tissue of these patients as well. This is in partial contradiction to the proposed shift of the macrophage phenotype from M1 to M2 in metabolically healthy obese patients. However, a general

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reduction of infiltrating macrophages might have beneficial effects on the release of vasoactive substances.

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A very interesting finding is the increased expression of adiponectin in the same amount of perivascular adipose tissue of internal mammary arteries of obese patients with a BMI> 30 kg/m2. In contrast, the leptin expression was not changed in the same perivascular adipose tissue. Several studies support differential roles of adiponectin and leptin in the cardiovascular

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system [21]. Leptin plasma levels have been shown to correlate with the amount of adipose tissue while adiponectin plasma levels were downregulated in obesity [5, 21]. Dysfunctional adipocytes produce less adiponectin. Oxidative stress reduces the production of adiponectin as

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well [5]. The local upregulation of adiponectin expression in the perivascular adipose tissue might reflect a healthy adipocyte phenotype and a compensatory mechanism to preserve

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endothelial function. A growing body of evidence supports a vasoprotective role of adiponectin [5]. Adiponectin has been shown to modulate macrophage function in the direction of an anti-inflammatory phenotype [22]. Adiponectin inhibits the macrophage-tofoam cell transformation by suppression of class A scavenger receptors [23]. Overproduction of adiponectin inhibits atherosclerotic plaque formation and inhibits class A scavenger receptor expression. However, experimental studies modulating the adiponectin expression in atherosclerotic mouse models did not affect plaque formation [24]. Therefore, additional

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studies are necessary to define the role of adiponectin in endothelial function and atherosclerosis [5]. It has been recently suggested that adiponectin plays a crucial role in preserving endothelial function in diabetic patients. Adiponectin was shown to suppress the NADPH oxidase activity

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in the vessel wall of diabetic and non-diabetic patients [25]. Hypoadiponectinemia is a key feature in the development of the vascular complications of type 2 diabetes [25]. However, diabetic patients have lower serum adiponectin levels in comparison to healthy patients [26].

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Higher adiponectin concentration might contribute to a better preservation of endothelial function and vasodilation.

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Finally, with respect to a role of adiponectin in vessel preservation during cold storage during transplantation procedures we have previously reported that endothelial injury may be partly prevented by addition of adiponectin [2]. This may support graft patency to avoid atherosclerosis-associated impairment of perfusion.

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We did observe differences in noradrenaline-mediated contraction (Fig. 1D), expression of integrin alpha X chain (Fig. 3A), mannose receptor 1 (Fig. 3B), and leptin (Fig. 3D) in perivascular adipose tissue while analyzing all patients or non-diabetic patients only. This

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supports slight differences in contractility and infiltration of perivascular adipose tissue by macrophages in non-diabetic patients compared to all patients. The increased leptin

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expression in perivascular adipose tissue further supports a beneficial adipokine profile in non-diabetic patients with a BMI >30 kg/m2. All other results were not different between both patient groups.

Differences in antihypertensive (or statin) therapy could affect vascular function by changing the concentration or response to vasoactive substances (e.g. NO, angiotensin II). However, we did not observe significant differences in the pharmacological therapy of our patients groups with different BMI (see Tab. 1). This further supports a specific impact of the BMI on the observed changes.

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Strength of our study is the analysis of the vascular function in clinical samples of patients with coronary artery disease. These studies need a specialized logistics and a close collaboration between experts from cardiac surgery, physiology and vascular biology. The role of the perivascular adipose tissue in vascular function is not well-understood and

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currently worldwide under intense investigation. Weaknesses of our study are the limited number of patients, the analysis of basic vascular function and the currently missing data on vascular function with perivascular adipose tissue. These new studies were recently approved

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by the ethics committee and will be the focus of future investigations.

In conclusion, our data suggest a reduced endothelial function in internal mammary arteries of

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adipose patients with a BMI between 25 and 30 kg/m2, compared to lean and obese patients. The group of obese patients studied (BMI above 30 kg/m2) also exhibited reduced infiltration of macrophages in the perivascular adipose tissue and increased adiponectin expression which

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may have contributed to the maintenance of endothelial function in these patients.

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ACCEPTED MANUSCRIPT Acknowledgements

This work was supported by funding of the Excellence Initiative by the German Federal and State Governments (Institutional Strategy, measure "support the best" to H.M. and A.D.), by grants from Else Kröner-Fresenius-Stiftung (Grant 2010_A105 to H.M.), Doktor Robert

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Pfleger-Stiftung (H.M.), and Deutsche Forschungsgemeinschaft (DFG) (Grant MO 1695/4-1, 1695/5-2 to H.M.). We are grateful to Jennifer Mittag and Melanie Brux for excellent

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technical assistance.

Disclosure

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MC, HL, BZ, CB, AD, KM and HM do not report disclosures.

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S, Funahashi T, Matsuzawa Y. Adipocyte-derived plasma protein, adiponectin, suppresses lipid accumulation and class A scavenger receptor expression in human monocyte-derived macrophages, Circulation, 2001;103:1057-63. [24] Nawrocki AR, Hofmann SM, Teupser D, Basford JE, Durand JL, Jelicks LA, Woo CW,

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Kuriakose G, Factor SM, Tanowitz HB, Hui DY, Tabas I, Scherer PE. Lack of association between adiponectin levels and atherosclerosis in mice, Arterioscler. Thromb. Vasc. Biol., 2010;30:1159-65. 10.1161/ATVBAHA.109.195826

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[25] Antonopoulos AS, Margaritis M, Coutinho P, Shirodaria C, Psarros C, Herdman L, Sanna F, De Silva R, Petrou M, Sayeed R, Krasopoulos G, Lee R, Digby J, Reilly S,

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Bakogiannis C, Tousoulis D, Kessler B, Casadei B, Channon KM, Antoniades C. Adiponectin as a link between type 2 diabetes and vascular NADPH oxidase activity in the human arterial wall: the regulatory role of perivascular adipose tissue, Diabetes, 2015;64:2207-19. 10.2337/db14-1011

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[26] Tong HV, Luu NK, Son HA, Hoan NV, Hung TT, Velavan TP, Toan NL. Adiponectin and pro-inflammatory cytokines are modulated in Vietnamese patients with type 2

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diabetes mellitus, J Diabetes Investig, 2016. 10.1111/jdi.12579

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Table 1: Clinical and study characteristics of all and non-diabetic patients with different body-mass index undergoing coronary artery bypass grafting surgery used for determination of vascular function BMI 25-30

BMI >30

P-value

all/non-diab.

all/non-diab.

all/non-diab.

all/non-diab.

Number (n)

18/13

25/20

24/9

Age

69/66

66/66

67/67

0.540/0.993

Ejection fraction

55%/54%

53%/56%

58%/54%

0.504/0.885

Smoking status

39%/54%

44%/35%

38%/44%

0.894/0.581

28%/0

20%/0

62%/0

0.013*/-

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Diabetes mellitus type 2

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BMI <25

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Characteristics

100%/100%

96%/95%

100%/100%

0.438/0.588

Dyslipidemia

44%/62%

72%/65%

71%/67%

0.128/0.968

ACE inhibitors

50%/69%

60%/70%

42%/44%

0.450/0.393

AT1 receptor

33%/15%

20%/15%

33%/44%

0.516/0.174

89%/92%

88%/95%

83%/78%

0.848/0.349

72%/77%

80%/85%

83%/89%

0.685/0.747

89%/92%

92%/95%

88%/78%

0.876/0.349

33%/31%

44%/25%

67%/67%

0.083/0.089

Statins Aspirin

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Diuretics

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β-blockers

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Hypertension

BMI, body-mass index; ACE, angiotensin-converting enzyme; AT1 receptor, angiotensin II receptor type 1, non-diab., non-diabetic.

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ACCEPTED MANUSCRIPT Table 2. PCR primers. Primer

Sequence, 5‘-3‘

ACTA2

sense

CTCCCAGAATCCTGTGAAGCA

Alpha-actin 2

antisense

AAAACAGCCCTGGGAGCATC

sense

TCCTCACTTCCATTCTGACTGC

antisense

GTAGAACAGCTCCCAGCAACA

antisense

TTCGCAGCTATTAATGACACCC

ITGAX

sense

GGAGCTCCCGGTGAAGTATG

Integrin alpha X

antisense

TATTGACCTGGTATCTGTGCATGG

LEP

sense

CAAGCTGTGCCCATCCAAAAA

Leptin

antisense

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Gene

ADIPOQ

MRC1 antisense Mannose receptor 1

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sense

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Adiponectin

TGAAGTCCAAACCGGTGACT

GCGATTAATAACAGCTAGTGGAAG TTCTCCATAAGCCCAGTTTTCA CTTTTATCCAACAATCTCCTGGTTCTC

POLR2A

sense

ACCTGCGGTCCACGTTGTGT

RNA polymerase 2A

antisense

CCACCATTTCCCCGGGATGCG

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antisense

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ACCEPTED MANUSCRIPT Figures legends

Fig. 1. Vessel function. A) Left internal mammary artery with perivascular adipose tissue after coronary artery bypass grafting (CABG) surgery. B) Human skeletonized internal mammary artery. C) Vessel wall tension after contraction with potassium chloride in

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segments of internal mammary arteries from lean (BMI <25 kg/m2), adipose (BMI 25-30 kg/m2) or obese (BMI >30 kg/m2) patients (I) or non-diabetic patients (II) undergoing CABG surgery. D) Vessel wall tension after contraction with noradrenaline in arterial segments of all

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patients (I) or non-diabetic patients (II). E) Dose-response curves in response to acetylcholine (Ach) of all patients (I) or non-diabetic patients (II). F) LogEC50 for groups of all patients (I)

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or non-diabetic patients (II) with different BMI. G) Smooth muscle-dependent maximal relaxation in response to sodium nitroprusside (SNP) of all patients (I) or non-diabetic patients (II). *P<0.05 vs. indicated study group.

Fig. 2. Vascular structure and morphology. Cross sections of representative internal mammary

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arteries of patients with A) BMI < 25 kg/m2, B) BMI 25-30 kg/m2, or C) BMI > 30 kg/m2 after Elastica van Gieson staining. D) Relative media area in vessel rings of all patients (I) or non-diabetic patients (II) with different BMI normalized to total vessel area. E) Intima/media

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ratio in vessel rings of all patients (I) or non-diabetic patients (II) with different BMI. F)

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Expression of vascular smooth muscle cell marker alpha-actin 2 (ACTA2) in vessels of all patients (I) or non-diabetic patients (II) with BMI of <25, 25-30 or > 30 kg/m2. Values are given as mean±SD.

Fig. 3. Gene expression in perivascular adipose tissue of internal mammary arteries of all patients (I) or non-diabetic patients (II) with BMI <25, 25-30, or >30 kg/m2. Expression of A) Integrin alpha X chain (ITGAX/CD11c) as a marker of M1 macrophages, B) macrophage mannose receptor 1 (MRC1/CD206) as marker of M2 macrophages, C) adiponectin (ADPQ) and D) leptin (LEP) in perivascular adipose tissue of all patients (I) or non-diabetic patients

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(II) with a BMI <25, 25-30, or > 30 kg/m2. Values are given as mean±SD. *P<0.05 vs.

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indicated group of patients.

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25

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