A Human Ex Vivo Atherosclerotic Plaque Model to Study Lesion Biology

Summary

Atherosclerosis is a chronic inflammatory process. This manuscript illustrates an easy to use ex vivo model to investigate fresh carotid or coronary artery plaques. The ex vivo model allows for the investigation of potential substances on the inflammatory milieu in human atherosclerotic lesions and results can be analyzed by various methods.

Abstract

Atherosclerosis is a chronic inflammatory disease of the vasculature. There are various methods to study the inflammatory compound in atherosclerotic lesions. Mouse models are an important tool to investigate inflammatory processes in atherogenesis, but these models suffer from the phenotypic and functional differences between the murine and human immune system. In vitro cell experiments are used to specifically evaluate cell type-dependent changes caused by a substance of interest, but culture-dependent variations and the inability to analyze the influence of specific molecules in the context of the inflammatory compound in atherosclerotic lesions limit the impact of the results. In addition, measuring levels of a molecule of interest in human blood helps to further investigate its clinical relevance, but this represents systemic and not local inflammation. Therefore, we here describe a plaque culture model to study human atherosclerotic lesion biology ex vivo. In short, fresh plaques are obtained from patients undergoing endarterectomy or coronary artery bypass grafting and stored in RPMI medium on ice until usage. The specimens are cut into small pieces followed by random distribution into a 48-well plate, containing RPMI medium in addition to a substance of interest such as cytokines or chemokines alone or in combination for defined periods of time. After incubation, the plaque pieces can be shock frozen for mRNA isolation, embedded in Paraffin or OCT for immunohistochemistry staining or smashed and lysed for western blotting. Furthermore, cells may be isolated from the plaque for flow cytometry analysis. In addition, supernatants can be collected for protein measurement by ELISA. In conclusion, the presented ex vivo model opens the possibility to further study inflammatory lesional biology, which may result in identification of novel disease mechanisms and therapeutic targets.

Introduction

Atherosclerosis as a chronic inflammatory disease is one of main causes of death in industrialized nations^1-2. Complications of atherosclerosis, especially acute coronary syndromes, have been linked to rupture of vulnerable lesions, causing atherothrombosis and vessel occlusion³. Innate and adaptive immunity seem to be involved during all steps of atherogenesis^2,4-5. Although significant progress has been made in the treatment of myocardial infarction, effective prevention of atherosclerosis and adverse cardiovascular events are still unresolved. Thus, studying lesional biology is essential for increasing our knowledge on the pathophysiology of atherosclerosis and to allow identification of novel therapeutic targets and development of novel therapies.

In many cases, murine models are used to investigate the pathophysiology of specific diseases. However, studying atherogenesis using mouse models is accompanied by several limitations: (1) Usually, atherosclerotic mice receive a high cholesterol diet. The cholesterol levels in these models cannot be compared with those in patients with elevated cholesterol serum levels⁶. (2) There are substantial differences between the murine and human immune system; thus foxp3 is a specific marker of murine regulatory T cells, whereas human foxp3 expression in human T cells does not necessarily confer a regulatory phenotype⁷. Also, the Th1/Th2 paradigm as defined in humans is not fully transferable to murine T cells. (3) A number of markers that are used to identify murine monocytes and macrophages such as F4/80 and markers of classical (M1) vs. alternative (M2) activation patterns does not exist in human myeloid cells⁸. (4) Gene expression of murine and human peripheral blood monocytes has been found to be substantially different⁹.

Thus, in order to increase our understanding of chronic inflammatory processes in human atherosclerosis, we need to make use of models working with human tissues, blood or cells. Here, we describe a model of human plaque tissue culture, which allows investigation of potential novel substances in the concept of human inflammatory lesional biology.

Protocol

1. Prepare medium as follows

1. Culture Medium: RPMI medium.
2. Add 10% fetal calf serum (FCS).
3. Add 100 U/ml penicillin G, and 100 g/ml streptomycin.

2. Storage of fresh plaque cylinder until use

1. The carotid endarterectomy operation of patients with or without ischemic symptoms (stroke, transitory ischemic attack) with a significant carotid artery stenosis will be done by vascular surgeons and coronary artery endarterectomy during coronary artery bypass grafting by heart surgeons. Carotid / coronary plaques need to be removed en bloc to preserve the plaque structure as described previously⁵.
2. After plaque extirpation place the specimen in a medium filled tube and store it on ice (plaque needs to be completely covered with medium) until use.

3. Plaque processing

1. Use an adequate cell culture dish (e.g. 60 mm) and add 5 ml RPMI medium.
2. Place the plaque tissue into the culture dish (plaque should be completely covered with medium).
3. Hold the plaque tissue carefully at the edges of the tissue by using sterile forceps.
4. Cut off the edges of the plaque sample by using a sterile scalpel.
5. Divide plaque tissue in half.
6. Assess the lesion morphology macroscopically (calcified, lipid rich, ruptured, thrombus, fibrosis).
7. Analyze the exact plaque morphology after the ex vivo experiment by immunohistochemistry. Use the AHA classification ¹⁰.
8. Discard plaques with severe calcification or fibrosis.
9. Cut the plaque tissue into homologous small pieces (3 x 3 x 3 mm).
10. Shock freeze two plaque pieces and store them in liquid nitrogen for basic values of the lesion until use.
11. Prepare a 48-well plate.
12. Add 500 µl RPMI medium to each well used for the experiment.
13. Please use at least two plaque pieces for each group.
14. Add the substance of interest (e.g. specific cytokines and chemokines).
15. Use unstimulated plaque pieces as controls.
16. Randomly plant the appropriated number of plaque pieces into the wells.
17. Culture the plaque pieces for indicated time points.
18. For the plaque tissue stimulation experiment with Lipopolysaccharide (LPS)
    1. Use the following time points: 3 hr, 8 hr and 24 hr.
    2. Use 2 plaque pieces for the LPS and two for the unstimulated group for each time point respectively.
    3. Add 1 µg/ml of Lipopolysaccharide.
19. During the incubation, maintain the 48-well plate at 37 °C in humidified air containing 5% CO₂.

4. After indicated time harvest plaque tissue pieces

1. Shock freeze the plaque pieces for mRNA isolation and cDNA synthesis (for detailed information-see protocol section 5).
2. Collect the supernatant and stored it at -20 °C for ELISA analysis.
3. For western blotting, smash and lyse plaque tissue. Filter the lysate through a 0.65 µm and 0.1 µm centrifugal filter device.
4. Embed plaque tissue in tissue-tec or paraffin for immunohistochemistry stainings.

5. RNA extraction from cultured plaque pieces

1. Use a TissueLyser for homogenization.
2. Isolate the RNA by using the kit (see materials table) according to manufacturer's instructions.
3. Determine the RNA quantity and quality of the samples with spectrophotometer.
4. Use the Boehringer cDNA kit for reverse transcription according to manufacturer's instructions.
5. For quantitative PCR, use for instance the Roche real-time PCR kit with SYBR Green.

Results

Here we present a number of figures that demonstrate results of the ex vivo plaque culturing. To assess changes in the inflammatory milieu in response to the agent of interest in the ex vivo model experiment, we measure different molecules which are known to be primarily involved in atherogenesis. As representative pro-atherogenic cytokines we choose TNFa, IL6 and IFNg^2,11. In addition, we use von Willebrand factor and tissue factor to evaluate pro-thrombotic changes. Furthermore, to address ...

Discussion

Here we present an ex vivo plaque culture model to investigate the influence of potentially relevant substances on atherosclerotic lesion biology. The major advantage of this ex vivo method is the ability to evaluate the influence of indicated substances on inflammatory cells and their cellular interplay as well as inflammatory pathways and cascades within human atherosclerotic lesions. Several usable methods (e.g. RT-PCR, western blot, immunohistochemistry, flow cytometry, ELISA) help to provi...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
RPMI medium	Gibco	21875-091
FCS	Gibco	10270-106
Penicillin-streptomycin	Sigma	P-4458
15 ml Tube	Sarstedt	62,554,502
Culture dish (60 mm)	Orange Scientific	5550200
LPS	Sigma	L4516
Cell culture plates 48-well	Greiner	677102
Scalpel - single use	Feather	FEA200130011
TissueLyser	Precellys 24 Dual	Cat. No. EQ03119.200.RD010.0
RNeasy (Mini) Kit	Qiagen	Cat. No. 74104
Boehringer cDNA kit	Roche Diagnostics	Cat. No. 11483188001
Nanodrop spectrophotometer	Thermo Fisher Scientific