Atherosclerosis Methods and Protocols

Dedication
Preface
Contents
Contributors
Part I: Atherosclerosis: Pathogenesis, Model Systems and Emerging Concepts
	Chapter 1: Atherosclerosis: Pathogenesis and Key Cellular Processes, Current and Emerging Therapies, Key Challenges, and Futur...
		1 Introduction
		2 Atherogenesis
		3 Foam Cell Formation
		4 Plaque Progression and Rupture
		5 Current and Emerging Therapeutic Strategies
		6 Challenges of Atherosclerosis Treatment and Research
		7 Future Research Directions
		References
	Chapter 2: Key Roles of Inflammation in Atherosclerosis: Mediators Involved in Orchestrating the Inflammatory Response and Its...
		1 Introduction
		2 Inflammation in Atherosclerosis
			2.1 Role of IL-1β and the Inflammasome
		3 Therapeutic Approaches Targeting Hyperlipidemia
		4 Targeting the IL-1/6 Pathway of Inflammation
		5 Other Anti-inflammatory Agents Under Investigation
		6 Other Potential Avenues
		7 Challenges and Future Perspectives
		References
	Chapter 3: Survey of In Vitro Model Systems for Investigation of Key Cellular Processes Associated with Atherosclerosis
		1 Introduction
		2 Endothelial Cell Dysfunction
		3 Changes in Monocyte Function in Relation to Atherosclerosis
		4 Changes in Macrophage Function in Relation to Atherosclerosis
		5 Changes in Vascular Smooth Muscle Cell Function in Relation to Atherosclerosis
		6 Conclusions
		References
	Chapter 4: Survey of Approaches for Investigation of Atherosclerosis In Vivo
		1 Introduction
		2 Animal Models of Atherosclerosis
		3 Approaches for Investigation of Plasma/Blood Parameters Together with Plaque Burden and Cellular Content
		4 Other Approaches in Investigation of Atherosclerosis In Vivo
		5 Conclusions
		References
	Chapter 5: Exploring the Associations Between Clonal Hematopoiesis of Indeterminate Potential, Myeloid Malignancy, and Atheros...
		1 Introduction
		2 CHIP in Hematologic Malignancy
		3 CHIP and Atherosclerosis
		4 Future Directions
			4.1 Experimental Models of CHIP
			4.2 How Should Information on CHIP in the Clinic Be Used to Predict and Treat Hematological Malignancy and Cardiovascular Dise...
			4.3 Proof-of-Concept Therapeutic Targeting of CHIP
			4.4 Implications of CHIP for Other Tissues
		References
	Chapter 6: Peptides as Therapeutic Agents for Atherosclerosis
		1 Introduction
		2 HDL and Atherosclerosis
		3 Apolipoproteins and the Amphipathic Helix Theory
		4 Apolipoprotein Mimetic Peptides That Support the Amphipathic Helix Theory
		5 Platform for the Design of Amphipathic Helical Peptides
		6 First In Vivo Experiment Showing Inhibition of Atherosclerosis
		7 Studies in Several Laboratories
		8 Phase I Studies of ApoA-I Mimetic Peptide
		9 Design and Studies of apoE Mimetics
		10 Conclusions
		References
Part II: In Vitro and Ex Vivo Model Systems for Atherosclerosis Research
	Chapter 7: Isolation and Culturing of Primary Mouse and Human Macrophages
		1 Introduction
		2 Materials
			2.1 Murine Bone Marrow-Derived Macrophages
			2.2 Human Monocyte-Derived Macrophages
		3 Methods
			3.1 Isolation, Culture, and Polarization of Murine Bone Marrow-Derived Macrophages
			3.2 Isolation and Culture of Human Monocyte-Derived Macrophages
		4 Notes
		References
	Chapter 8: Effective Transfection and Gene Silencing of Primary Murine Macrophages with Small Interfering RNA
		1 Introduction
		2 Materials
			2.1 Production of L929 Cell Conditioned Medium
			2.2 Isolation, Culturing, and Differentiation of Primary Macrophages
			2.3 Transfection of Cells
		3 Methods
			3.1 Production of L929 Cell Conditioned Medium
			3.2 Isolation, Culturing and Differentiation of Primary Murine Bone Marrow-Derived Macrophages (BMDM) (See Note 5)
			3.3 Transfection of Cells
			3.4 Posttransfection
		4 Notes
		References
	Chapter 9: Monitoring Cellular Proliferation, Migration, and Apoptosis Associated with Atherosclerosis Plaques In Vitro
		1 Introduction
		2 Materials
			2.1 Proliferation: BrdU
				2.1.1 General Cell Culture
				2.1.2 Cell Culture: BrdU Incorporation
				2.1.3 Immunocytochemistry for BrdU
				2.1.4 Imaging and Analysis
			2.2 Proliferation: EdU
				2.2.1 Cell Culture: EdU Incorporation
				2.2.2 Fluorescent Labeling of EdU
				2.2.3 Imaging and Analysis
				2.2.4 Automated Data Analysis
			2.3 Proliferation: Western Blotting for Cell Cycle Markers
				2.3.1 Protein Extraction
				2.3.2 Protein Assay
				2.3.3 1D Polyacrylamide Gel Electrophoresis
				2.3.4 Stain-Free Total Protein Imaging
				2.3.5 Protein Transfer
				2.3.6 Immunodetection
				2.3.7 Densitometry
			2.4 Migration: Scratch Wound Assay
			2.5 Migration: Cell Exclusion Assay
			2.6 Apoptosis: Cleaved Caspase-3 Immunocytochemistry
				2.6.1 Immunocytochemistry for Cleaved Caspase-3
		3 Methods
			3.1 Cell Propagation from Frozen Stocks
			3.2 Proliferation: BrdU
				3.2.1 Cell Culture
				3.2.2 Immunocytochemistry for BrdU
				3.2.3 Imaging and Analysis
			3.3 Proliferation: EdU
				3.3.1 Cell Culture
				3.3.2 Fluorescent Labeling of EdU
				3.3.3 Imaging and Analysis
				3.3.4 Automated Data Analysis
			3.4 Proliferation: Western Blotting for Cell Cycle Proteins
				3.4.1 Cell Culture
				3.4.2 Protein Extraction
				3.4.3 Protein Assay
				3.4.4 1D Polyacrylamide Gel Electrophoresis
				3.4.5 Stain-Free Total Protein Imaging
				3.4.6 Protein Transfer
				3.4.7 Immunodetection
				3.4.8 Densitometry
			3.5 Migration: Scratch Wound Assay
				3.5.1 Cell Seeding
				3.5.2 Cell Treatment
				3.5.3 Image Analysis
			3.6 Migration: Cell Exclusion Assay
				3.6.1 Cell Seeding
				3.6.2 Removal of Culture Insert
				3.6.3 Imaging and Analysis
			3.7 Apoptosis: Cleaved Caspase-3 Immunofluorescence
				3.7.1 Cell Seeding
				3.7.2 Cell Treatment
				3.7.3 Cell Fixation
				3.7.4 Immunodetection
				3.7.5 Imaging Analysis
		4 Notes
		References
	Chapter 10: Monocyte Adhesion Assays for Detecting Endothelial Cell Activation in Vascular Inflammation and Atherosclerosis
		1 Introduction
		2 Materials
			2.1 Endothelial Cell Culture
			2.2 Human Peripheral Blood Mononuclear Cell (PBMC) Isolation
			2.3 Monocyte-Endothelial Cell Adhesion Assay
		3 Methods
			3.1 Preparation of Endothelial Cell Monolayer
			3.2 Preparation of Human Peripheral Blood Mononuclear Cells (PBMC)
			3.3 Monocyte-Endothelial Cell Adhesion Assay
			3.4 Example of Results
		4 Notes
		References
	Chapter 11: Chick Embryo Ex Vivo Aortic Sprouting Assays for Cardiovascular Research
		1 Introduction
		2 Materials
		3 Methods
			3.1 Prenatal Assessment of Fertilized Brown Leghorn Chick Embryo (Gallus gallus domesticus)
			3.2 Chick Aorta Dissection and Histological Analysis
			3.3 Chick Aortic Ring Assay
			3.4 Sprouting of Endothelial Cells: Zonal Segmentation and Generation Study
		4 Notes
		References
	Chapter 12: Purification and Analysis of Circulating Lipid Particles
		1 Introduction
		2 Materials
			2.1 Blood Taking and Plasma Purification
			2.2 Ultracentrifugation
			2.3 Agarose Gel Electrophoresis
			2.4 SDS-PAGE
			2.5 Protein and Cholesterol Assay
			2.6 Lipid Particle Labeling with Fluorescent DiI
			2.7 Cell Culture and Cell Lysis
			2.8 Confocal Microscopy
		3 Methods
			3.1 Blood Collection
			3.2 Lipid Particle Purification
			3.3 Lipid Particle Electrophoretic Analysis
				3.3.1 Agarose Gel Electrophoresis
				3.3.2 Protein Analysis of Lipid ParticlesLipid particles Using Gradient SDS-PAGE
			3.4 Cholesterol Assay of Lipid Particle Fractions
			3.5 Lipid Particle Labelling with Fluorescent DiI Derivatives
			3.6 HUVEC and HEK293T Cell Culture and Stimulation of DiI-Labeled Lipoprotein Uptake
			3.7 Confocal Microscopy
		4 Notes
		References
	Chapter 13: Preparation of LDL, Oxidation, Methods of Detection, and Applications in Atherosclerosis Research
		1 Introduction
		2 Materials
			2.1 Collection of Plasma and Isolation of Human LDL
			2.2 Isolation of LDL
			2.3 LDL Dialysis, Storing of LDL and Determination of Protein Concentration
			2.4 Preparation of Copper-Oxidized LDL or HDL
			2.5 Preparation of Myeloperoxidase (MPO)-oxidized LDL or HDL
			2.6 Preparation of Acetyl LDL
			2.7 Electrophoretic Mobility of Lipoproteins on Agarose  Gels
			2.8 Mouse Peritoneal Macrophage Isolation
			2.9 THP-1 Cell Culture
			2.10 Foam Cell Preparation
			2.11 Staining of Foam Cells
				2.11.1 Oil Red O Staining for Cultured Cells
				2.11.2 Nile Red Staining
			2.12 Macrophage Cholesterol Determination
			2.13 Reverse Cholesterol Transport Assay
			2.14 SDS-PAGE Gel Electrophoresis
		3 Methods
			3.1 Collection of Plasma
			3.2 Isolation of LDL
			3.3 LDL Dialysis, Storage, and Protein Concentration
			3.4 Preparation of Copper-Oxidized LDL/HDL
			3.5 Preparation of Myeloperoxidase (MPO)-Oxidized LDL/HDL
			3.6 Preparation of Acetyl LDL
			3.7 Electrophoretic Mobility of Lipoproteins on Agarose Gel
			3.8 Mouse Peritoneal Macrophage Isolation for Foam Cell Formation
			3.9 Cell Culture
				3.9.1 THP-1 Cell Culture
				3.9.2 Differentiation of Monocytes into Macrophages
			3.10 Preparation of Foam Cells
			3.11 Staining of Foam Cells
				3.11.1 Oil Red O Staining for Cultured Cells
				3.11.2 Nile Red staining
			3.12 Macrophage Cholesterol Determination
			3.13 Reverse Cholesterol Transport Assay
			3.14 SDS-PAGE Gel Electrophoresis
		4 Notes
		References
	Chapter 14: Monitoring Modified Lipoprotein Uptake and Macropinocytosis Associated with Macrophage Foam Cell Formation
		1 Introduction
			1.1 OxLDL Uptake
			1.2 Macropinocytosis
		2 Materials
			2.1 General Reagents and Equipment for oxLDL Uptake and Macropinocytosis Assays
			2.2 Reagents for oxLDL Uptake Assay
			2.3 Reagents for Macropinocytosis
		3 Methods
			3.1 OxLDL Uptake Assay
			3.2 Lucifer Yellow-Based Macropinocytosis Assay
		4 Notes
		References
	Chapter 15: A Fluorescence-Based In Vitro Method to Assess Cholesterol Efflux
		1 Introduction
		2 Materials
			2.1 Growth Period
			2.2 Labeling Period
			2.3 Equilibration Period
			2.4 Acceptors Period
			2.5 Determination of Cholesterol Content in the Media and Cells
		3 Methods
			3.1 Growth Period: Cells Thaw, Growth and Seeding
			3.2 Labeling Period: Labeling Cells with Fluorescent Cholesterol
			3.3 Equilibration Period: Cholesterol Equilibration and Cell Conditioning
			3.4 Acceptors Period: Promotion of ChE
			3.5 Determination of Cholesterol Content in the Media and Cells
			3.6 Calculation of Acceptors-Mediated ChE
			3.7 Monitoring Fluorescence of the Labeling Media and the Experimental Conditions
		4 Notes
		References
	Chapter 16: Monitoring the Anisotropy and Fluidity of the HDL Monolayer as Surrogates of HDL Functionality
		1 Introduction
		2 Materials
		3 Methods
		4 Notes
		References
	Chapter 17: Assessment of Ex Vivo Antioxidative Potential of Murine HDL in Atherosclerosis
		1 Introduction
		2 Materials
		3 Methods
			3.1 Human LDL Isolation
			3.2 Murine HDL Isolation
			3.3 Oxidation Assay
		4 Notes
		References
	Chapter 18: Dual-Fluorescence In Vitro Efferocytosis Assay
		1 Introduction
		2 Materials
			2.1 Culturing of L929 Cells for L929-Conditioned Media (L9CM)
			2.2 Culturing of the Jurkat T Cell  Line
			2.3 Bone Marrow-Derived Macrophage (BMDM) Isolation and Culture
			2.4 Apoptotic Cell Preparation, Fluorescence Labeling, and Biotinylation
			2.5 Efferocytosis Assay
		3 Methods
			3.1 Culturing of L929 Cells for L929-Conditioned Media (L9CM)
			3.2 Culturing of the Jurkat T Cell  Line
			3.3 BMDM Isolation and Culture
			3.4 Apoptotic Cell Preparation, Fluorescence Labeling, and Biotinylation
			3.5 Efferocytosis Assay
		4 Notes
		References
	Chapter 19: Investigation of Mitochondrial Bioenergetic Profile and Dysfunction in Atherosclerosis
		1 Introduction
		2 Materials
		3 Method
			3.1 Cell Preparation (Prior to the Assay)
			3.2 Assay Preparation (The Day Before the Assay)
			3.3 Setting Up the Software (The Day of the Assay)
			3.4 Preparation of Drug Compounds
			3.5 Assay
		4 Notes
		References
	Chapter 20: Probing Inflammasome Activation in Atherosclerosis
		1 Introduction
			1.1 Enzyme-Linked Immunosorbent Assay (ELISA)
			1.2 Western Blotting
		2 Materials
			2.1 Cell Culture
			2.2 ELISA
			2.3 Western Blotting
		3 Methods
			3.1 Cell Priming and Stimulation
			3.2 ELISA
				3.2.1 Day Prior to Assay
				3.2.2 ELISA
			3.3 Western Blotting
				3.3.1 Cell Lysis and Protein Extraction
				3.3.2 Sample Preparation for SDS-PAGE
				3.3.3 Loading and Electrophoresis of the Gel for SDS-PAGE
				3.3.4 Transferring the Protein from the Gel to the Membrane
				3.3.5 Antibody Staining
		4 Notes
		References
	Chapter 21: Investigation of Platelet Aggregation in Atherosclerosis
		1 Introduction
		2 Materials
			2.1 Preparation of Platelet-Rich Plasma (PRP) and Platelet-Poor Plasma (PPP)
			2.2 Platelet Agonist Preparation
		3 Methods
			3.1 Preparation of Platelet-Rich Plasma (PRP) and Platelet-Poor Plasma (PPP)
			3.2 Basic Aggregometry Protocol
			3.3 Application of LTA for the Evaluation of Platelet Reactivity of PRP Induced by Inflammatory and Thrombotic Mediators and P...
			3.4 LTA for the Evaluation of the Inhibitory Effects (IC50 Values) of an Antiplatelet Agent Against Platelet Aggregation of  P...
		4 Notes
		References
	Chapter 22: Extracellular Vesicles in Atherosclerosis Research
		1 Introduction
		2 Materials
			2.1 Turbidity and Lysis Assay: EV and Coagulation
			2.2 EV and Monocyte Migration
			2.3 EV and Thrombus Formation
			2.4 EV and Leukocyte Adhesion to Endothelial Cells
		3 Methods
			3.1 Turbidity and Lysis Assay: EV and Coagulation
			3.2 EV and Monocyte Migration
			3.3 EV and Thrombus Formation
			3.4 EV and Leukocyte Adhesion to Endothelial Cells
		4 Notes
		References
	Chapter 23: The Use of Wire Myography to Investigate Vascular Tone and Function
		1 Introduction
		2 Materials
		3 Methods
			3.1 Dissection of Small Mesenteric Resistance Vessels
			3.2 Mounting the Mesenteric Resistance Vessels
			3.3 Equilibration and Normalization
			3.4 Assessing if the Vessel Is Viable
			3.5 Calculating the EC50 for Mesenteric Vessels in Response to U46619
			3.6 Assessment of Endothelial Integrity
			3.7 Concentration-Response Curve to Acetylcholine to Assess vasodilatory Response
			3.8 Cleaning Procedure for Myograph Chamber Following Experiments
			3.9 Data Analysis
		4 Notes
		References
Part III: In Vivo Model Systems for Atherosclerosis Research
	Chapter 24: Pig and Mouse Models of Hyperlipidemia and Atherosclerosis
		1 Introduction
		2 Hyperlipidemia and Atherosclerosis in the  Pig
			2.1 Pig Lipoproteins
			2.2 Pig Models of Atherosclerosis
				2.2.1 Diet-Induced Atherosclerosis
				2.2.2 Natural and Genetically Engineered Mutant  Pigs
				2.2.3 Atherosclerosis Regression
		3 Hyperlipidemia and Atherosclerosis in the Mouse
			3.1 Mouse Lipoproteins
			3.2 Mouse Models of Atherosclerosis
				3.2.1 Genetically Engineered Mouse Models of Atherosclerosis
				3.2.2 Immune Cells in Atherosclerotic Models
				3.2.3 Human apoE Isoforms Expressed in the Mouse
				3.2.4 ApoE Expression in Hepatocytes and Other Cells
				3.2.5 Additional Ldlr Deficient Models
				3.2.6 Coronary Artery Atherosclerosis
				3.2.7 The Intestinal Microbiome
				3.2.8 Regression of Atherosclerosis
		4 Conclusion
		References
	Chapter 25: Use of Rabbit Models to Study Atherosclerosis
		1 Introduction
		2 Materials
			2.1 Preparation of a Cholesterol Diet
			2.2 Collection of Blood
			2.3 Preparation and Analysis of Plasma Lipids and Lipoproteins
			2.4 Rabbit Autopsy and Separation of Aortas
			2.5 Sudan IV Staining and Aortic Gross Lesion Analysis
			2.6 Dissecting the Aorta for Sections
			2.7 Histological and Immunohistochemical Staining
			2.8 Quantification of Microscopic Lesion Area and Macrophage/Smooth Muscle Cells
		3 Methods
			3.1 Preparation of a Cholesterol Diet
			3.2 Collection of Blood
			3.3 Preparation of Plasma and Analysis of Lipids and Lipoproteins
			3.4 Rabbit Autopsy and Collection of Aortas
			3.5 Staining Aortas with Sudan IV Stain
			3.6 Dissection of the Aorta and Preparation of Paraffin Sections
			3.7 Histological and Immunohistochemical Staining
			3.8 Quantitation of Microscopic Lesions and Cellular Components
		4 Notes
		References
	Chapter 26: Genetically Engineered Hamster Models of Dyslipidemia and Atherosclerosis
		1 History of Hamster Use in Lipid Metabolism Disorders and Atherosclerosis Studies
		2 Transgenic Hamsters
		3 Gene Knockout Hamsters
			3.1 LDLR Knockout Hamsters
				3.1.1 The Application Value of Heterozygous LDLR Knockout Hamsters in Drug Development
			3.2 LCAT Knockout Hamsters
			3.3 ABCA1 Knockout Hamsters
			3.4 ApoC2 Knockout Hamsters
			3.5 ApoC3 Knockout Hamsters
		4 Limitations of Hamsters as Animal Models of Dyslipidemia and Atherosclerosis
		References
	Chapter 27: Atherosclerosis Induced by Adeno-Associated Virus Encoding Gain-of-Function PCSK9
		1 Introduction
		2 Materials
			2.1 rAAV-PCSK9DY Production
				2.1.1 Plasmid Transfection
				2.1.2 Harvest of the Cell Lysate
				2.1.3 Purification in an Iodixanol Gradient
				2.1.4 rAAV-PCSK9DY Particle Concentration and Suspension
			2.2 Tail Vein Injection of the rAAV-PCSK9DY Solution
		3 Methods
			3.1 rAAV-PCSK9DY Production (See Note 2)
				3.1.1 Plasmid Transfection
				3.1.2 Harvest of the Cell Lysate
				3.1.3 Purification in an Iodixanol Gradient
				3.1.4 rAAV-PCSK9DY Concentration and Titration
			3.2 Tail Vein injection of the rAAV-PCSK9DY Solution (See Notes 9 and 10)
		4 Notes
		References
	Chapter 28: Monitoring Leukocyte Migration During Atherosclerosis In Vivo
		1 Introduction
		2 Materials
			2.1 Fixation and Sectioning
			2.2 Immunostaining
		3 Methods
			3.1 Formalin Fixation, Sectioning of Paraffin-Embedded Aorta, and Deparaffinization
			3.2 CD11b Immunostaining
		4 Notes
		References
	Chapter 29: Evaluation of Plaque Burden and Lipid Content in Atherosclerotic Plaques
		1 Introduction
			1.1 Hematoxylin and Eosin (H&E)
			1.2 Oil Red O
			1.3 Masson´s Trichrome
		2 Materials
			2.1 Mouse Dissection
			2.2 En Face Staining
			2.3 Sectioning
			2.4 General Staining
			2.5 H&E Staining
			2.6 ORO Staining
			2.7 Masson´s Trichrome Staining
		3 Methods
			3.1 Mouse Dissection
			3.2 En Face Preparation and Staining of the Aorta
			3.3 Sectioning Using a Cryostat
			3.4 H&E Staining
			3.5 ORO Staining
			3.6 Masson´s Trichrome Staining
			3.7 Image Analysis Using ImageJ
		4 Notes
		References
	Chapter 30: Monitoring Cellularity and Expression of Key Markers in Atherosclerotic Plaques
		1 Introduction
		2 Materials
		3 Method
			3.1 Staining
			3.2 Image Analysis
		4 Notes
		References
	Chapter 31: Monitoring Cellular Proliferation and Apoptosis in Atherosclerosis Plaques and Intimal Thickenings
		1 Introduction
		2 Materials
			2.1 Immunohistochemistry for Markers of Proliferation and Apoptosis, and BrdU
				2.1.1 Deparaffinization and Rehydration
				2.1.2 Antigen Retrieval
				2.1.3 Quenching Endogenous Peroxidase Activity (for Colorimetric Detection Only)
				2.1.4 DNA Denaturation (for BrdU and EdU Detection Only)
				2.1.5 Protein Blocking
				2.1.6 Immunodetection
				2.1.7 Mounting
				2.1.8 Imaging and Analysis
			2.2 Fluorescent Labeling of EdU in Tissue Sections
				2.2.1 Deparaffinization and Rehydration
				2.2.2 Antigen Retrieval
				2.2.3 Detection of EdU
				2.2.4 Mounting, Imaging, and Analysis
		3 Methods
			3.1 Immunohistochemistry for Markers of Proliferation and Apoptosis, and BrdU
				3.1.1 Deparaffinization and Rehydration
				3.1.2 Antigen Retrieval
				3.1.3 Quenching Endogenous Peroxidase Activity
				3.1.4 DNA Denaturation
				3.1.5 Protein Blocking
				3.1.6 Immunodetection
				3.1.7 Mounting
				3.1.8 Imaging and Analysis
			3.2 Fluorescent Labeling of EdU in Tissue Sections
				3.2.1 Deparaffinization and Rehydration
				3.2.2 Antigen Retrieval
				3.2.3 Detection of EdU
				3.2.4 Mounting, Imaging, and Analysis
		4 Notes
		References
	Chapter 32: Investigation of Atherosclerotic Plaque Vulnerability
		1 Introduction
		2 Materials
			2.1 Chemicals
				2.1.1 Picro-Sirius Red (PSR) Staining
				2.1.2 Elastin Van Gieson (EVG) Staining
				2.1.3 Oil Red O (ORO) Staining
				2.1.4 Immunohistochemistry for Macrophages and Vascular Smooth Muscle Cells
			2.2 Equipment
				2.2.1 PSR Staining
				2.2.2 EVG Staining
				2.2.3 ORO Staining
				2.2.4 Immunohistochemistry
		3 Methods
			3.1 Picro-Sirius Red Histochemistry for Fibrillar Collagens
			3.2 Elastin Van Gieson Histochemistry for Elastin
			3.3 Oil Red O Histochemistry for Neutral Lipids
			3.4 Immunohistochemistry for Macrophages and Vascular Smooth Muscle Cells
			3.5 Identification of Buried Fibrous Layers
			3.6 Assessment of Plaque Vulnerability
		4 Notes
		References
	Chapter 33: Use of Mouse Carotid Artery Ligation Model of Intimal Thickening to Probe Vascular Smooth Muscle Cell Remodeling a...
		1 Introduction
		2 Materials
			2.1 Surgical Procedure
				2.1.1 Materials
				2.1.2 Equipment (See Fig. 3)
			2.2 Sample Preservation, Processing, and Analysis
				2.2.1 Materials
					Formalin-Fixed Samples (See Note 3)
					Isopentane-Frozen Samples (See Note 4)
					Liquid Nitrogen Frozen Samples (See Note 5)
					Elastic Van Gieson (EVG) Staining
					Immunodetection of BrdU Incorporation
				2.2.2 Equipment
					Formalin-Fixed Samples
					Isopentane-Frozen Samples
					Liquid Nitrogen Frozen Samples
					EVG Staining
					Immunodetection of Incorporated BrdU
		3 Methods
			3.1 Surgical Procedure
				3.1.1 Carotid Artery Ligation
				3.1.2 Termination of Animal and Collection of Arterial Samples
			3.2 Preservation of Samples
				3.2.1 Formalin-Fixation of Samples
				3.2.2 Isopentane-Frozen Samples
				3.2.3 Frozen Samples
			3.3 Processing of Arterial Samples
				3.3.1 Formalin-Fixed Samples
				3.3.2 Isopentane-Frozen Samples
				3.3.3 Liquid Nitrogen Frozen Samples
					Protein Extraction
					mRNA Extraction
			3.4 Histological Analysis
				3.4.1 Elastin Van Gieson Staining (See Note 25)
				3.4.2 Immunodetection of BrdU Incorporation (See Notes 33 and 34)
					Deparaffinization and Rehydration
					Antigen Retrieval and Quenching of Endogenous Peroxidase
					DNA Denaturation (See Note 40)
						Immunohistochemistry (Colorimetric)
						Immunofluorescence
					Protein Blocking
					Immunodetection
					Mounting
					Imaging and Analysis
		4 Notes
		References
	Chapter 34: Assessing Efferocytosis in Atherosclerotic Lesions In Situ.
		1 Introduction
		2 Materials
			2.1 Rehydration of Paraffin-Embedded Tissue Sections on Glass Sides
			2.2 Antigen Retrieval
			2.3 TUNEL Reaction and Anti-Mac2 Immunostaining
		3 Methods
			3.1 Rehydration of Tissue Sections on Glass Sides
			3.2 Antigen Retrieval
			3.3 TUNEL Reaction and Immunostaining with an Anti-Mac2 Antibody
		4 Notes
		References
	Chapter 35: Monitoring Atheroprotective Macrophage Cholesterol Efflux In Vivo
		1 Introduction
		2 Materials
			2.1 Preparation of Acetylated LDL (ac-LDL)
			2.2 Cell Culture
			2.3 Macrophage Radiolabeling and Injection
			2.4 Quantification of Macrophage Cholesterol Efflux
		3 Methods
			3.1 Acetylation of LDL
			3.2 Cell Culture
			3.3 Macrophage Radiolabeling and Injection
			3.4 Sample Collection and Quantification of Macrophage Cholesterol Efflux and Overall Transport to Feces In Vivo
		4 Notes
		References
	Chapter 36: Flow Cytometry Analysis of Hematopoietic Stem/Progenitor Cells and Mature Blood Cell Subsets in Atherosclerosis
		1 Introduction
		2 Materials
			2.1 Cell Harvesting and Processing
				2.1.1 Bone Marrow (BM) and Spleen
				2.1.2 Peripheral Blood (PB)
			2.2 Cell Staining
				2.2.1 General Requirements for Staining
				2.2.2 Hematopoietic Stem Cells, Primitive Progenitors, and Committed Progenitors from  BM
				2.2.3 Mature Hematopoietic Cells in BM, PB, and Spleen
			2.3 Cell Analysis and Characterization Using Flow Cytometry
		3 Methods
			3.1 Sample Harvesting and Processing
				3.1.1 PB
				3.1.2 BM and Spleen
			3.2 Cell Staining
				3.2.1 Hematopoietic Stem Cells and Primitive Progenitors from the BM
				3.2.2 Committed Hematopoietic Progenitors from the BM
				3.2.3 Hematopoietic Mature Cells in BM, PB, and Spleen
				3.2.4 Control Samples
			3.3 Flow Cytometry Analysis
		4 Notes
		References
	Chapter 37: Whole Mount Preparation of Mouse Aorta for Confocal Microscopy Studies of the Intima
		1 Introduction
		2 Materials
		3 Methods
			3.1 Perfusion Fixation of Mouse Aorta
			3.2 Dissection of the Mouse Aorta and Heart
			3.3 Aorta Processing
			3.4 Immunofluorescence Staining
			3.5 Confocal Imaging of the Aortic Intima Layer
			3.6 Image Analysis of the Aortic Intima Layer
		4 Notes
		References
	Chapter 38: Isolation of Mouse Aortic RNA for Transcriptomics
		1 Introduction
		2 Materials
			2.1 Mouse Aorta Dissection and Collection of Intima, Media, and Adventitia
			2.2 RNA Extraction
		3 Method
			3.1 Mouse Aorta Dissection
				3.1.1 Intima Collection
				3.1.2 Media and Adventitia Collection
			3.2 RNA Isolation
				3.2.1 RNA Isolation from Whole Mouse Aorta
				3.2.2 RNA Isolation from Intima
				3.2.3 RNA Isolation from Media and Adventitia
		4 Notes
		References
	Chapter 39: Redox Proteomics Analysis of Atherosclerotic Aortas: Application of the ``OxICAT´´ Method
		1 Introduction
			1.1 Tissue Homogenization
			1.2 Preventing Artificial Oxidation
			1.3 OxICAT
			1.4 LC-MS/MS Analysis and Data Processing
		2 Materials
		3 Methods
			3.1 Tissue Homogenization
			3.2 Preventing Artificial (Ex Vivo) Oxidation and Induce Thiol Trapping
			3.3 OxICAT
				3.3.1 Labeling with Cleavable ICAT Reagents
				3.3.2 Trypsin Digestion
				3.3.3 Cation Exchange for Cleaning Up the Peptides
				3.3.4 Avidin Affinity Chromatography for Peptide Purification
				3.3.5 Biotin Cleavage
			3.4 LC-MS/MS Analysis and Data Processing
		4 Notes
		References
	Chapter 40: Intravital Microscopy in Atherosclerosis Research
		1 Introduction
		2 Materials
			2.1 Multiphoton Microscope
			2.2 Exogeneous Fluorophores
			2.3 Murine Model
			2.4 Epifluorescence Microscope
		3 Methods
			3.1 Mouse Preparation
			3.2 Thrombus Model
			3.3 Multiphoton Microscopy
			3.4 MPM Image Analysis
			3.5 Correlative Fluorescence Microscopy
		4 Notes
		References
	Chapter 41: Localization of Long Noncoding RNA in Formalin-Fixed, Paraffin-Embedded Vascular Tissue Using In Situ Hybridization
		1 Introduction
		2 Materials
			2.1 Equipment
			2.2 Reagents
		3 Methods
			3.1 Slide Preparation
			3.2 Antigen Retrieval
			3.3 Probe Hybridization
			3.4 Stringency Washes
			3.5 Detection
			3.6 Counterstain and Fixation
		4 Notes
		References
Part IV: Emerging Techniques in Atherosclerosis Research
	Chapter 42: In Vivo Gene Editing in Lipid and Atherosclerosis Research
		1 Introduction
			1.1 AAV-CRISPR for Editing Candidate Genes in the Liver
			1.2 AAV-CRISPR for Modeling Hypercholesterolemia and Atherosclerosis
		2 Materials
			2.1 General Materials and Equipment
			2.2 gRNA Design
			2.3 Plasmid Construction
			2.4 In vivo Study
			2.5 Analysis of On-Target and Off-Target Nuclease Activity
			2.6 Evaluation of Gene Knockdown by Western Blot
		3 Methods
			3.1 gRNA Design
				3.1.1 Target Gene Annotation
				3.1.2 gRNA Design-Visual Selection
				3.1.3 Off-Target Prediction Using COSMID
				3.1.4 gRNA Design and Off-Target Prediction Using CRISPOR
			3.2 Plasmid Construction and AAV Production
				3.2.1 Oligonucleotides Annealing
				3.2.2 Cloning and Analysis
				3.2.3 AAV Packaging
			3.3 In vivo Study
				3.3.1 AAV Preparation and Injection
				3.3.2 Bleeding and Plasma Isolation
				3.3.3 Liver Dissection
			3.4 Analysis of On-Target and Off-Target Nuclease Activity
				3.4.1 Liver DNA Isolation and Quantification
				3.4.2 Primers Design for Sanger Sequencing-Based Methods
				3.4.3 TIDE and ICE Analyses
				3.4.4 Targeted Deep Sequencing
				3.4.5 Integration PCR
				3.4.6 AAV Genome PCR
			3.5 Evaluation of Gene Knockdown by Western Blot
		4 Notes
		References
	Chapter 43: Laser Capture Microdissection-Based mRNA Expression Microarrays and Single-Cell RNA Sequencing in Atherosclerosis ...
		1 Introduction
		2 Materials
			2.1 Data Acquisition
			2.2 Data Analysis
		3 Methods
			3.1 Microarray Data Analysis
				3.1.1 Microarray Data Preprocessing
				3.1.2 Determination of DEGs and Inflammation-Related GO Terms
			3.2 scRNA-seq Data Analysis
				3.2.1 scRNA-seq Data Preprocessing
				3.2.2 Determination of DEGs and Inflammation-Related GO Terms
			3.3 Profiling Cellular Abundance in Microarray  Data
			3.4 Comparing the Results Obtained from Microarray Analysis and scRNA-Seq Analysis
		4 Notes
		References
	Chapter 44: Combined Single-Cell RNA and Single-Cell α/β T Cell Receptor Sequencing of the Arterial Wall in Atherosclerosis
		1 Introduction
		2 Materials
			2.1 Aorta Dissection and Single-Cell Suspension Preparation
			2.2 Single-Cell Library Construction
				2.2.1 Equipment and Reagents
				2.2.2 PCR Reaction Mixture
			2.3 Sequencing
			2.4 Data Analysis
			2.5 Mice
		3 Methods
			3.1 Dissection of Atherosclerotic Plaques and ATLOs from the Diseased Aorta
			3.2 Preparation of Single-Cell Suspension of Plaques, ATLOs, and LNs
			3.3 Single-Cell Sorting
			3.4 Gel Bead Emulsions (GEMs) Generation and Reverse Transcription
			3.5 5′ Gene Expression Library Construction
			3.6 TCR Enrichment and Library Construction
			3.7 Sequencing
			3.8 Data Analysis
				3.8.1 Sequencing Alignment
				3.8.2 Single Cell Transcriptome Analysis in RStudio
				3.8.3 TCR Repertoire Analysis in RStudio
				3.8.4 Combined Transcriptome and TCR Repertoire Analysis in RStudio
		4 Notes
		References
	Chapter 45: Tissue Clearing Approaches in Atherosclerosis
		1 Introduction
		2 Materials
			2.1 Mice
			2.2 Reagents
				2.2.1 Perfusion and Fixation Reagents
				2.2.2 Decolorization and Permeabilization Reagents
				2.2.3 Antibodies
				2.2.4 Clearing Reagents
			2.3 Equipment and Other Material
				2.3.1 Imaging Microscopes
				2.3.2 Image Analyses Tools
		3 Methods
			3.1 Whole-Body Immunostaining and 3DISCO Tissue Clearing
				3.1.1 Tissue Preparation
				3.1.2 Tissue Clearing
				3.1.3 Imaging 3DISCO Cleared Tissue
			3.2 Whole-Mount Imaging of Isolated Aorta and TDE Clearing
				3.2.1 Tissue Preparation
				3.2.2 Tissue Clearing
				3.2.3 Imaging of TDE Cleared Sample Using Multiphoton Laser Scanning Microscope (MPLSM)
				3.2.4 Imaging of TDE Cleared Sample Using  CLSM
			3.3 Image Processing and Analyses
				3.3.1 Image Processing of Light-Sheet Images
				3.3.2 Image Processing of MPLSM and CLSM Images
		4 Notes
		References
	Chapter 46: Single-Cell Antibody Sequencing in Atherosclerosis Research
		1 Introduction
			1.1 The Development of Single-Cell Technology
			1.2 Why Surface Phenotype Matters
			1.3 Assessing Cell Surface Phenotype
				1.3.1 TotalSeq
				1.3.2 AbSeq
			1.4 Challenges
			1.5 Flow of Antibody Sequencing and Analysis
		2 Materials
			2.1 Materials for both TotalSeq and AbSeq
			2.2 Materials for TotalSeq
			2.3 Materials for AbSeq
		3 Methods
			3.1 TotalSeq
				3.1.1 Viability Check
				3.1.2 Labeling with Sample Tags and Mixing
				3.1.3 Antibody Staining
			3.2 AbSeq
				3.2.1 Viability Check
				3.2.2 Labeling with Sample Tags and Mixing
				3.2.3 Antibody Staining
		4 Notes
		References
	Chapter 47: Flow Cytometry and Mass Cytometry for Measuring the Immune Cell Infiltrate in Atherosclerotic Arteries
		1 Introduction
		2 Materials
			2.1 Mouse aorta Harvesting
			2.2 Aorta Digestion
			2.3 Flow Cytometry (FACS)
			2.4 Mass Cytometry (CyTOF)
		3 Methods
			3.1 Mouse Aorta Harvesting
			3.2 Aorta Digestion
			3.3 Flow Cytometry (FACS)
				3.3.1 FACS Panel Design
				3.3.2 FACS Extracellular Staining
				3.3.3 FACS Sample Acquisition
				3.3.4 FACS Data Analysis
			3.4 Mass Cytometry (CyTOF)
				3.4.1 CyTOF Panel Design
				3.4.2 CyTOF Internal Controls (See Notes 17-20)
				3.4.3 CyTOF Antibody Cocktails (See Note 21)
				3.4.4 CyTOF Live/Dead Staining
				3.4.5 CyTOF Cell Staining
				3.4.6 CyTOF Cell Fixation
				3.4.7 CyTOF Intercalator Stain and Intracellular Staining
				3.4.8 CyTOF Acquisition
				3.4.9 CyTOF Cleaning Data
				3.4.10 CyTOF Analysis
		4 Notes
		References
	Chapter 48: Contrast-Enhanced Ultrasound Molecular Imaging in Atherosclerosis Research
		1 Introduction
		2 Materials
		3 Methods
			3.1 Biotinylated Microbubble Preparation
			3.2 Microbubble Conjugation
			3.3 Surgery for Intravenous Injection in Anesthetized  Mice
			3.4 CEU Molecular Imaging
			3.5 Image Analysis
		4 Notes
		References
	Chapter 49: Nanoprobes for Computed Tomography and Magnetic Resonance Imaging in Atherosclerosis Research
		1 Introduction
		2 Materials
		3 Methods
			3.1 Preparation of Lipid Anchored Targeting Ligand
			3.2 Preparation and Characterization of CT Nanoprobes
				3.2.1 Preparation of Iodixanol Solution (10 mL, 400 mg Iodine/mL)
				3.2.2 Preparation of Liposomal-Iodine CT Nanoprobe Formulations
				3.2.3 Determination of Iodine Content in CT Nanoprobe Formulations
			3.3 Preparation and Characterization of MR Nanoprobes
				3.3.1 Preparation of Liposomal-Gadolinium MR Nanoprobe Formulations
				3.3.2 Determination of Gadolinium and Lipid Content in MR Nanoprobes
			3.4 In Vivo MR Imaging
			3.5 In Vivo CT Imaging
		4 Notes
		References
	Chapter 50: Positron Emission Tomography in Atherosclerosis Research
		1 Introduction
		2 Materials
		3 Methods
			3.1 In Vivo PET/CT of Small Animals
			3.2 Ex Vivo Digital Autoradiography
			3.3 Clinical PET
		4 Notes
		References
	Chapter 51: Three-Dimensional Visualization of Atherosclerotic Vessels by Tissue Clearing and Light-Sheet Fluorescence Microsc...
		1 Introduction
		2 Materials
			2.1 Vessel Harvesting and Fixation
			2.2 Adipo-Clear
			2.3 Light Sheet Fluorescence Microscopy
			2.4 Image Processing and Analysis
		3 Methods
			3.1 Vessel Harvesting and Fixation
			3.2 Adipo-Clear
			3.3 Light Sheet Fluorescence Microscopy
			3.4 Image Processing and Analysis
		4 Notes
		References
	Chapter 52: Intravascular Fluorescence Molecular Imaging of Atherosclerosis
		1 Introduction
		2 Materials
			2.1 NIRF-OCT Imaging System and Catheter
			2.2 OCT Console
			2.3 NIRF Console
			2.4 NIRF-OCT Catheter
			2.5 Dual-Channel Rotary Junction
			2.6 NIRF Calibration and Data Processing
			2.7 Experimental Rabbit Atherosclerosis Model
			2.8 Angioplasty Balloons
			2.9 Intravascular Ultrasound Imaging
			2.10 NIRF-OCT Imaging
			2.11 Molecular Analysis of Tissue
			2.12 Histology
		3 Methods
			3.1 Experimental Atherosclerosis Lesion Generation in Rabbits
			3.2 Randomization of Angioplasty Therapy
			3.3 Intravascular Ultrasound (IVUS) Imaging
			3.4 Intravascular NIRF-OCT Imaging of Plaque Inflammation and Structure
			3.5 Randomization of Angioplasty Therapy: Experimental Setup
			3.6 Sacrifice and End of Study: Experimental Setup
			3.7 Molecular Analysis: Aortic Tissue
			3.8 Molecular Analysis: RNA Extraction and cDNA Synthesis
		4 Notes
		References
Index