Quantitative Analysis of Cellular Composition in Advanced Atherosclerotic Lesions of Smooth Muscle Cell Lineage-Tracing Mice

Summary

We propose a standardized protocol to characterize the cellular composition of late-stage murine atherosclerotic lesions including systematic methods of animal dissection, tissue embedding, sectioning, staining, and analysis of brachiocephalic arteries from atheroprone smooth muscle cell lineage tracing mice.

Abstract

Atherosclerosis remains the leading cause of death worldwide and, despite countless preclinical studies describing promising therapeutic targets, novel interventions have remained elusive. This is likely due, in part, to a reliance on preclinical prevention models investigating the effects of genetic manipulations or pharmacological treatments on atherosclerosis development rather than the established disease. Also, results of these studies are often confounding because of the use of superficial lesion analyses and a lack of characterization of lesion cell populations. To help overcome these translational hurdles, we propose an increased reliance on intervention models that employ investigation of changes in cellular composition at a single cell level by immunofluorescent staining and confocal microscopy. To this end, we describe a protocol for testing a putative therapeutic agent in a murine intervention model including a systematic approach for animal dissection, embedding, sectioning, staining, and quantification of brachiocephalic artery lesions. In addition, due to the phenotypic diversity of cells within late-stage atherosclerotic lesions, we describe the importance of using cell-specific, inducible lineage tracing mouse systems and how this can be leveraged for unbiased characterization of atherosclerotic lesion cell populations. Together, these strategies may assist vascular biologists to more accurately model therapeutic interventions and analyze atherosclerotic disease and will hopefully translate into a higher rate of success in clinical trials.

Introduction

Atherosclerosis is the leading cause of morbidity and mortality worldwide underlying the majority of coronary artery disease, peripheral artery diseases, and stroke. Late-stage coronary atherosclerosis can lead to severe complications including myocardial infraction accounting for nearly 16% of world population mortality^1,2. Due to its devastating impact on public health, substantial effort has been made to decipher the mechanisms driving atherosclerosis progression, as well as to develop novel therapeutic strategies. Yet, the Likelihood of Approval (LOA) rate of clinical trials for cardiovascular disease is a....

Protocol

Animal breeding, handling and procedures were approved by the University of Virginia and the University of Pittsburgh Institutional Animal Care and Use Committee.

1. Generation of SMC lineage tracing mice

1. Breed Myh11-Cre/ERT2 males²⁸ (Jackson Laboratory; #019079) with R26R-EYFP females (Jackson Laboratory; #006148) to obtain Myh11-Cre/ERT2⁺ R26R-EYFP^+/+ males.
2. Breed the Myh11-Cre/ERT2⁺ R26R-EYFP^+/+ males with ApoE^-/- female mice (Jackson laboratory; #002052). Cross the progeny and select by genotyping Myh11

Results

Myh11-Cre/ERT2 R26R-EYFP Apoe^-/- mice were injected with tamoxifen between six and eight weeks of age before being fed a high fat diet. At 18 weeks of high fat diet feeding, two groups of eight mice were treated weekly with either a mouse monoclonal anti-IL-1β antibody or an isotype-matched IgG control at 10 mg/kg for 8 weeks (Figure 1)⁷. Mice were sacrificed and perfused with a 4% PFA-PBS solution. Brachiocephalic .......

Discussion

Despite decades of research and technical advances in studying atherosclerosis, the field has a disappointing history of translating scientific findings to clinical therapies^34,35. This phenomenon may be explained in part by discrepancies in animal models, experimental designs, and lesion analyses. Herein, we describe an experimental pipeline that we used to analyze the cellular composition in advanced atherosclerotic lesions using lineage tracing mice

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
16% Paraformaldehyde aqueous solution	Electron Microscopy Sciences	RT 15710	Tissue perfusion and fixation
23G butterfly needle	Fisher	BD367342
25G needle	Fisher	14-821-13D
A1 Confocal microscope	Nikon		Confocal microscope
ACTA2-FITC antibody (mouse)	Sigma Aldrich	F3777	Primary Antibody
Alexa-647 anti goat	Invitrogen	A-21447	Secondary antibody
Antigen Unmasking solution, Citric acid based	Vector Labs	H-3300	Antigen retrieval solution
Chow Diet	Harlan Teklad	TD.7012
Coverslip	Fisher	12-544-14	Any 50 x 24 mm cover glass
DAPI (4',6-Diamidino-2-Phenylindole, Dihydrochloride)	Invitrogen	D1306	Nucleus fluorescent counterstaining
Donkey Alexa-488 anti-rabbit	Invitrogen	A-21206	Secondary antibody
Donkey Alexa-555 anti-rat	Abcam	ab150154	Secondary antibody
DPBS 10X without Calcium and Magnesium	Gibco	14200166	PBS for solution dilutions and washes. Dilute to 1x in deionized water
Embedding cassette	Fisher	15-182-701D
ETDA vacuum tube	Fisher	02-685-2B
Ethanol 200 proof	Decon	2701
Foam pad	Fisher	22-222-012
Gelatin from cold water fish skin	Sigma Aldrich	G7765
GFP antibody (goat)	abcam	ab6673	Primary antibody
goat IgG control	Vector Labs	I-5000	IgG control
High Fat Diet	Harlan Teklad	TD.88137
ImageJ	NIH		Computer program https://imagej.nih.gov/ij/
LGALS3 antibody (rat)	Cedarlane	CL8942AP	Primary antibody
LSM700 confocal microscope	Zeiss		Confocal microscope
Microscope Slides, Superfrost Plus	Fisher	12-550-15
Microtome blades	Fisher	30-538-35
Mouse IgG control	Vector Labs	I-2000	IgG control
NIS element imaging software	Nikon		Imaging software for z-stack image acquisition
Normal Horse serum	Sigma Aldrich	H1270
Pap Pen	Fisher	50-550-221
Peanut oil	Sigma	P2144
Prolong gold Antifade mountant	Invitrogen	P36930	Mounting medium
Rabbit IgG control	Vector Labs	I-1000	IgG control
Rat IgG control	Vector Labs	I-4000	IgG control
RUNX2 antibody (rabbit)	Abcam	ab192256	Primary Antibody
Syringe	BD	309628	1 ml syringe
Tamoxifen	Sigma	T5648
Xylene	Fisher	X55K-4
Zen imaging software	Zeiss		Imaging software for z-stack image acquisition