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Perivascular adipose tissue (PAT) inflammation may have a role in coronary artery disease (CAD) pathophysiology. However, most evidence has come from samples obtained during surgical procedures that may imply in some limitations. Moreover, the role of B lymphocytes and inflammation in PAT that is adjacent to unstable atheroma plaques has not been investigated in humans using morphometric measurements.
The objective of this study is to investigate the inflammation in PAT, subcutaneous, and perirenal adipose tissues (SAT and PrAT) among chronic CAD, acute CAD, and control groups in an autopsy study.
Heart, SAT, and PrAT samples are collected from autopsied subjects in a general autopsy service, with the written informed consent of the next-of-kin (NOK). Sociodemographic and clinical data are obtained from a semistructure interview with the NOK. Coronary arteries are dissected and PAT are removed. Sections with the greatest arterial obstruction or unstable plaques, and the local with absence of atherosclerosis in all coronary arteries are sampled. PAT are represented adjacent to these fragments. Adipose tissues are fixed in 4% buffered paraformaldehyde solution and analyzed immunohistochemically for macrophages (CD68), macrophage polarization (CD11c for proinflammatory and CD206 for anti-inflammatory), B lymphocytes (CD20), and T lymphocytes (CD3). Slides will be scanned, and inflammatory cells will be quantified in 20 random fields. Participants will be categorized in CAD groups, after morphometric measurement of arterial obstruction and plaque composition analysis in accordance with American Heart Association classification. Three study groups will be investigated: acute CAD (at least one unstable plaque); chronic CAD (≥50% arterial obstruction); and controls (<50% arterial obstruction). Inflammatory cells in PAT, SAT, and PrAT will be counted and compared between groups using multivariate linear regression, adjusted for age, body mass index, hypertension, diabetes, alcohol use, and smoking.
We present the methods of our study that was developed from 2 pilots. Currently, data collection and tissue processing are ongoing. Data collection, histology and immunochemistry procedures, and quantification of all inflammatory cells are expected to be concluded within 1 year.
This study will contribute for the understanding of the mechanisms of CAD pathophysiology because it will help to clarify the role of inflammation both in chronic and acute CAD.
Cardiovascular disease is the leading global cause of death, accounting for 17.3 million deaths per year worldwide, and its prevalence is expected to grow to 23.6 million by 2030 [
Perivascular adipose tissue (PAT) surrounds most systemic blood vessels, except for the cerebral circulation; this may be a specialized type of adipose tissue related to inflammation and CAD severity [
The present manuscript aimed to describe standardized methods that were developed to investigate the association between inflammation of PAT and CAD in an autopsy study. The specifics aims of our study are to (1) investigate the association of macrophages, T and B lymphocytes with chronic CAD, acute CAD, and controls, (2) investigate the correlation between number of inflammatory cells in periplaque PAT and percentage of arterial obstruction, (3) investigate the association between number of inflammatory cells in periplaque PAT and atheroma plaque composition, and (4) compare the number of macrophages, polarized macrophages, T and B lymphocytes in periplaque PAT with the number of the same inflammatory cells in a control area far from the atheroma plaque, in SAT and in PrAT in the same individual.
This observational cross-sectional autopsy study was approved by the local ethics committee. Written informed consent is obtained from the next-of-kin (NOK) of all participants before starting any study procedure.
The Sao Paulo Autopsy Service (SPAS) is a general autopsy service based at the University of Sao Paulo that performs approximately 13,000 autopsies per year. In Sao Paulo city, autopsy is mandatory when an individual dies from a natural cause, and where the cause of death is unclear or the individual did not have medical assistance. Since 2004, the Brazilian Aging Brain Study Group has sourced autopsy material from SPAS to investigate the normal and pathological aging brain [
While the NOK waits for the autopsy of their deceased family member, they are invited to participate in this study. Nurses explain the aim of the study to the NOK and ask for their signature on an informed consent form. For all subjects to be included in this study, the NOK needs to have at least weekly interactions with the deceased and can therefore provide reliable information. A semistructured interview relating to sociodemographic and clinical information is conducted with each NOK prior to the collection of tissue samples (
Study outline. SPAS: Sao Paulo Autopsy Service; ICF: informed consent form; SAT: subcutaneous adipose tissue; PrAT: perirenal adipose tissue; PAT: perivascular adipose tissue.
The inclusion criteria for this study are: individuals of age ≥30 years at the time of death; a post mortem interval of ≤24 hours; a NOK with at least weekly contact with the deceased in the 6 months prior to death; and the availability of heart, SAT, and PrAT tissues from the medical pathologist. We included participants aged 30 years or older because advanced atherosclerotic plaques could be found in individuals with at least 30-years old at death [
The exclusion criteria employed in this study include: inconsistent clinical data provided by the NOK; adipose tissue fixation >72 hours; the use of corticosteroids or immunosuppressants, the presence of autoimmune disease, pericarditis, pericardial effusion, myocarditis, endocarditis, Chagas’ disease, hemopericardium, cardiac tamponade, coronary artery stent, or previous cardiac surgery. Patients with these conditions are excluded because they could induce or suppress inflammation in an atheroma plaque-independent manner. During the autopsy, samples of the major organs (heart, lung, spleen, kidney, and liver) are collected, processed, and stained with hematoxylin and eosin. Individuals that have an infection and show 2 or more criteria associated with sepsis in these samples, as defined previously [
The cause of death is determined by certified pathologists, based on the autopsy. In addition to the semistructured interview relating to the deceased’s sociodemographic information (including age, race, and education in years completed) [
The heart is collected to obtain PAT and coronary arteries. SAT is removed from the region of the umbilical scar and PrAT from the kidney. The heart, SAT, and PrAT is washed in running water to remove blood and clots.
Two processes were compared for heart fixation. The first involved fixation of the heart (with EAT) by immersion in 70% alcohol for at least 24 hours. After this period, we dissected the right coronary artery, left coronary artery trunk, anterior descending coronary artery, and circumflex coronary artery, together with 1 cm of the adjacent PAT and all EAT was removed from myocardium. PAT was removed systematically and cut into 1.5 cm sections, starting from the ostium (
Therefore, we changed our fixation protocol. The second fixation process involved the coronary artery dissection with the adjacent PAT immediately after heart collection to test whether the dissection of fresh coronary arteries and PAT would be more effective. The heart was incubated at −20°C for 2 minutes prior to injecting agar inside each coronary artery, and then the heart was placed at −20°C for another 2 minutes to solidify the agar. The coronary arteries were then dissected as described above and PAT was removed. The second fixation protocol has two advantages. First, the injection of agar before artery dissection allows agar infiltration until minor branches. Second, when we dissect the coronary arteries, there are already solidified agar inside the arterial lumen, facilitating arterial opening after repeated injections of agar (
We also investigated whether the 10% formaldehyde solution and 4% buffered paraformaldehyde solution (pH 7.2‑7.4) influenced coronary artery fixation. Coronary arteries samples were identified and then fixed for 5 days. We used the same fragment of coronary artery divided in 3 to compare agar injection procedures and fixatives. Agar prevented artery flattening (
We compared adipose tissue fixation times of 24, 48, and 72 hours in 4% buffered paraformaldehyde solution. Fixation times >72 hours were not tested because they can lead to a loss of antigenicity or the production of formic acid, which can impair identification of immune cells [
Primary antibodies used to identify inflammatory cells.
Target | Specification | Dilution | Inflammatory cell |
CD3 | Polyclonal rabbit anti-human | 1:1500 | T lymphocytes [ |
CD20 | Monoclonal mouse anti-human clone L26 | 1:12,000 | B lymphocytes |
CD68 | Monoclonal mouse anti-human clone KP-1 | 1:5000 | Macrophages [ |
CD11c | Monoclonal rabbit anti-human clone EP1347Y | 1:400 | Macrophages polarized M1 [ |
CD206 | Monoclonal mouse anti-human clone 5C11 | 1:1500 | Macrophages polarized M2 [ |
Perivascular adipose tissue (PAT) and coronary artery preparation of pilot 2. A: Agar was initially injected in the ostium of right coronary artery, the heart was cooled, and PAT was sampled at 1.5 cm intervals from the ostium until the final trajectory of the coronary artery. B: After PAT removal, the coronary artery was flat partially. C: The revised fixation procedure included at least two agar injections, and the coronary arteries were effectively open.
Fixation of the coronary artery. A: Coronary artery without agar injection and fixation in 10% formaldehyde solution. B: Coronary artery with agar injection and fixation in 10% formaldehyde solution. C: Coronary artery with agar injection and fixation in 4% buffered paraformaldehyde solution. D: Coronary artery flattening observed following heart fixation by immersion in alcohol prior to dissection of the coronary arteries (photographed macroscopically). E: Section of the same tissue shown in panel D, photographed microscopically after staining.
Immunochemistry of perivascular adipose tissue (PAT) using an anti-CD3 primary antibody. PAT was fixed for A: 24 hours; B: 48 hours; and C: 72 hours. Red arrows indicate CD3-positive T lymphocytes. All images were obtained using a microscope at 20× magnification.
Area measurements: the internal elastic lamina (IEL) and lumen. A: Area delineated by the IEL. B: Area of the lumen.
After fixation, coronary arteries are washed in running water for 30 minutes to remove excess paraformaldehyde solution. All coronary arteries are cut in 5-mm sections to identify the section with the largest obstruction or unstable plaques (eg, hemorrhage or thrombus), using a magnifying glass (Emporionet LP 500). An area without atherosclerosis that is distal to the section with the largest degree of arterial stenosis is also sampled. The number of atherosclerotic plaques is counted along the artery, as a measure of the extent of atherosclerosis. The agar is removed from inside the arterial lumen and the sections are photographed using a stereomicroscope (Nikon SMZ 1000).
The coronary arteries are decalcified, dehydrated, diaphonized, and immersed in paraffin prior to cutting 4 μm sections, using a microtome and staining with Verhoeff’s stain, as well as hematoxylin and eosin. The sections are then photographed using a stereomicroscope. The percentage of arterial obstruction is measured by morphometric methods using an image processing software (ImageJ). We measure the area of the lumen and the area delineated by the internal elastic lamina (IEL) (
After we complete the tissue processing, participants will be classified into 3 groups: chronic CAD, with ≥50% obstruction in at least one artery [
PAT, SAT, and PrAT are cut into 4 µm sections, applied to silanized slides (3-aminopropyltriethoxysilane), and immersed in paraffin. Prior to immunochemistry, the sections of PAT that correspond with sampled coronary artery fragments, SAT, and PrAT are deparaffinized by placing the slides in hot xylene in an oven at 60°C to 65°C for 5 minutes and then dipping in 3 baths of cold xylene. The sections are then hydrated in 95% alcohol, followed by 70% alcohol, washed in tap water and deionized water, and placed in phosphate buffer, pH 7.4. Antigen recovery is performed in 10 mM citric acid, pH 6, at a high temperature in a pressure cooker. Endogenous peroxidase is blocked using 3% hydrogen peroxide prior to incubating the slides with the indicated primary antibodies in the presence of 1% bovine serum albumin for 24 hours at 4°C. The slides are then incubated with the appropriate horseradish peroxidase-conjugated secondary antibody and EasyLink One and the signal is generated using the chromogen, diaminobenzidine. The sections are counterstained using Harris hematoxylin.
The slides are scanned and analyzed at 40× magnification using the Pannoramic Viewer software. This program hides the slide identification so that the operator is blinded to the subject’s diagnosis, the source and type of adipose tissue, and hotspots (ie, accumulation of inflammatory cells). Twenty random fields with 600 µm of diameter were also analyzed with no magnification (cells can only be observed with precision at 20× magnification). This process is systematic and large cell agglomerates (identified in >20× magnification) are avoided. Inflammatory cells are counted per the primary antibody staining and these results were expressed as the number of cells per micrometers squared.
To calculate the sample size, we used a previous study that found an effect size of 0.93, with a mean standard deviation of 44 (SD 21) inflammatory cells/µm² in a CAD group and 24 (SD 22) inflammatory cells/µm² in a control group [
Currently, data collection and tissue processing are ongoing. The data collection, histology and immunochemistry procedures, and quantification of inflammatory cells are expected to be concluded by May 2017.
Although EAT thickness and volume can be evaluated using imaging methods [
Although the association of CAD with macrophages and T lymphocytes has been investigated previously [
It is possible that inflammation in PAT could contribute locally to the development of the atherosclerotic plaque, as suggested by previous imaging [
PAT inflammation may be a measurable and modifiable risk factor that could be used in clinical practice. Some studies have investigated interventions that aim to reduce the inflammatory burden, for example by reducing the EAT volume via a reduction of the total body weight or by promoting the conversion of white adipose tissue into brown adipose tissue, which is associated with a decreased risk of obesity-related disorders. Other studies have investigated drugs that modulate immune receptors to reduce inflammation [
However, while inflammation and PAT thickness have been suggested to show positive associations with the degree of arterial stenosis [
Nevertheless, one limitation of the present study is that the sociodemographic and clinical information are collected after death from the NOK. To improve the accuracy of this information, we only include NOK with daily or weekly interactions with the deceased. Moreover, the reliability of the post mortem interview had been demonstrated by a previous study from our group, which showed a high sensitivity (87%) and specificity (94%) using this approach [
Here, we described the protocol that we are using to investigate the association between CAD and inflammation in adipose tissues. Particularly, we described in details the pilot studies that we performed to fixate and process the arteries and adipose tissues. These measures are important to allow for unbiased morphometric measures of atherosclerosis and inflammatory cell counting.
American Heart Association
analysis of the variance
coronary artery disease
Coordenação de Aperfeiçoamento de Pessoal de Nível Superior
epicardial adipose tissue
Fundação de Amparo à Pesquisa do Estado de São Paulo
informed consent
internal elastic lamina
next-of-kin
perivascular adipose tissue
perirenal adipose tissue
subcutaneous adipose tissue
Sao Paulo Autopsy Service
This work was supported by a grant from Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) [2013/00335-2]. DSFI received a scholarship from FAPESP and AN received a scholarship from Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES).
DSFI, CAP, and CKS were responsible for study design. DSFI and CKS contributed to manuscript writing. AN contributed to the method design of pilot 1. DSFI, CAP, and CKS were responsible for data analysis and interpretation. LFFS helped with standardization of the immunochemistry protocols and will evaluates signs of sepsis. FMC and KCSS helped to collect the samples. DSFI and FMC performed dissections. DSFI will evaluate coronaries and count immune cells. REPL, LTG, RELFR, and WJF contributed resources and tissue bank support. All authors reviewed the paper.
None declared.