Atherosclerosis on the ASK


Overview

Atherosclerosis is disease of the arterial blood vessels (arteries) characterized by the build-up of waxy plaques on the inside of blood vessels (in the inner, endothelial layer). It is a condition in which the artery wall thickens as a result of the buildup of fats and cholesterol in and on artery walls (plaques), which can restrict blood flow. These plaques can burst, triggering a blood clot (thrombosis) or, if the flow of blood is completely blocked, they could cause a heart attack via the coronary arteries or a stroke if the carotid arteries are affected.

Atherosclerosis is characterized by a chronic inflammatory response in the walls of arteries caused largely by the accumulation of macrophage white blood cells and promoted by low-density lipoproteins (LDL) without adequate removal of fats and cholesterol from the macrophages by functional high-density lipoproteins (HDL). Substances or processes that cause atherosclerosis are called atherogenic. It is caused by the formation of multiple plaques within the arteries. It is a chronic disease that commonly remains asymptomatic for decades.

The initial stages of atherosclerosis are characterized by infiltration of macrophages and growth of isolated foam cells. This leads to the development of “fatty streaks” in the arterial wall (small sub-endothelial deposits of monocyte-derived macrophages). Promoted by LDLs and inadequate HDLs this tissue insult drives an inflammatory cascade that recruits additional macrophages to the artery wall. The macrophages, which ingest oxidized LDL, slowly turn into large “foam cell” macrophages, containing numerous internal cytoplasmic vesicles with high lipid content. Death of these foam cells further propagates the inflammatory process.

ApoE-deficient (apoE-/-) mice model many aspects of the pathogenesis of human disease. When on high cholesterol diets, these mice develop sever hypercholesterolemia and lesions that progress from fatty streaks to fibrous plaques in lesion-prone areas throughout the aorta.


Products for imaging atherosclerosis

AgentAgent TypeAgent MechanismAvailable Wavelengths and optimal in vivo imaging time (post-injection)Route of metabolism/ background tissue(s)Validated imaging methods*
ProSense®Activatable; fluorescenceBroadly detects cathepsin activity which is upregulated in inflammatory cells such as foam cells. It is taken up in the lysosomes of inflammatory cells associated with atherosclerosis and cleaved by multiple cathepsins, activating fluorescent signal.680 nm (24-48 hours)

750 nm (FAST) (24 hours)

750 nm (6-24 hours)		Liver (680 nm)

Low liver/intestine (750 nm FAST)

Low liver/bladder (750 nm)		In vivo/Ex vivo, Flow cytometry, In vitro microscopy
Cat B FAST™Activatable; fluorescenceTaken up into the lysosomes of atherosclerosis-associated inflammatory cells and is selectively activated by cathepsin B.680 nm (36 h)

750 nm (36 h)		Salivary glands, liver, kidneysIn vivo/Ex vivo, Flow cytometry, In vitro microscopy
Cat K FAST™Activatable; fluorescenceSelectively activated by osteoclast-associated cathepsin K at sites of bone resorption or tissue calcification. This may be used in later stages of atherosclerosis development when calcification occurs.680 nm (6-24 h)Kidney, liverIn vivo/Ex vivo, Flow cytometry, In vitro microscopy
MMPSense™Activatable; fluorescenceMMP-2 and MMP-9 are considered important biomarkers in the formation of a vulnerable plaque.645 nm (6-24 h)

680 nm (24-36 h)

750 nm (12-24 h)		Liver and kidneys (645 nm and 750 nm)

Liver (680 nm)		In vivo/ex vivo
Annexin-Vivo™Targeted; fluorescenceTargeted probe that labels cells that are undergoing the early stages of apoptosis. It is useful in visualizing the progression of atherosclerosis as cellular necrosis occurs increasingly during later stages of the disease.750 nm (2 h)Kidneys (high), liverIn vivo/Ex vivo, Flow cytometry, In vitro microscopy
IntegriSense™Targeted; fluorescenceSmall molecule αvß3 integrin antagonist that contains a NIR fluorophore reporter. This targeted agent detects increased integrin expression associated with neovasculature, tumors, and some inflammatory cells associated with atherosclerosis.645 nm (48 h)

680 nm (24 h)

750 nm (24 h)		Bladder/kidneys (645 nm)

Kidneys/intestine (680 nm)

Kidneys (750 nm)		In vivo/Ex vivo, Flow cytometry, In vitro microscopy
OsteoSense®Targeted; fluorescenceBisphosphonate imaging agent that enables imaging of bone growth and resorption; can be used to measure bone cancer metastasis.680 nm (3-24 h)

750 nm (3-24 h)

800 nm (3-24 h)		BladderIn vivo
AngioSPARK®Vascular; fluorescencePegylated fluorescent nanoparticles that remain localized in the vasculature for extended periods of time and enable imaging of blood vessels and angiogenesis680 nm (0 - 4 hours)Long term tissue accumulationIn vivo

Choosing the right agent

When choosing an agent for angiogenesis studies, several factors should be taken into consideration:

  • Fluorescent vs. Bioluminscent agents: Be sure to choose the proper agent for your instrumentation. Some imagers can measure both bioluminescence and fluorescence, while others can only measure one or the other. 
  • Dye excitation/emission wavelength: Some fluorescent agents are available with excitation wavelengths that range from 645 nm to 800 nm. Be sure to pick a wavelength that is appropriate for both your instrument and application. Most microscopes filters are not suitable for wavelengths above 680 nm.  However deep tissue imaging typically has less background fluorescence and works better with longer wavelengths (750 nm- 800 nm). 
  • Agent clearance time is typically faster for activatable agents, which allows for re-injection after shorter time periods.  
  • Agent specificity: For targeted and activatable agents, it is important to remember that the protein or enzyme that the agent is targeting needs to be present and well expressed in your mouse model. 
  • In vivodistribution: Some agents might accumulate in organs at timepoints which could interfere with your study. Be sure to check the biodistribution profile of each agent of interest. 
  • Type of imaging required for your study: Vascular agents for example, while useful in assessing vascularity changes in the mouse, do not make good agents for in vitro cell imaging or tissue analysis. If these types of analyses are desired, targeted or activatable agents might be a better choice for you.


Model Systems/mouse models (Example Data)

Experimental Animals. Specific pathogen-free female Apolipoprotein (apo) E-deficient mice (6-8 weeks of age, 18–20 g) were obtained from The Jackson Labs (Bangor, ME) and housed in a controlled environment (72°F; 12:12-h light-dark cycle) under specific pathogen-free conditions. Mice homozygous for the Apoetm1Unc mutation show a marked increase in total plasma cholesterol levels that are unaffected by age or sex. Fatty streaks in the proximal aorta are found at 3 months of age, and the lesions increase with age. To accelerate the onset of disease, these mice were fed a 0.2% high cholesterol diet (Western type No. 88137, Harlan Laboratories, Madison WI) for assessment at different timepoints relative to the start of feeding (from 15 to 30 weeks on diet). Age-matched C57BL/6 mice were used as wild-type (WT) controls for all studies and were maintained on standard chow and water. All experiments were performed in accordance with VisEn Medical IACUC guidelines for animal care and use.

NIR Fluorescent Imaging Agents for the Detection ofAtherosclerosis. A suite of biomarker-specific VisEn imaging agents (Table 1) were used to detect atherosclerosis severity, progression, and response to therapy. Individual agents targeted at specific biomarkers of atherosclerotic disease were imaged using FMT quantitative tomography to quantify protease activity or tissue integrin levels in vivo. Each of the agents were administered intravenously in all imaging studies. The typical adult mouse dose is 2 nmoles in 150 μL saline, but for these studies a higher dose of 8 nmoles/mouse was used to enhance signal within the very small anatomical region of the aortic arch. Imaging of ProSense and IntegriSense was performed 24h following injection of the agent, whereas optimal imaging timepoints for the FAST agents (Cat K and Cat B FAST) was 6h.

atherosclerosis1.jpg
Figure 1. FMT 2500 images of ProSense 750 in ApoE-/- and WT Mice. A. Images from 5 representative apoE-/- mice maintained on high-cholesterol diets for 30 weeks were generated by FMT 2500 24h after ProSense 750 was injection (8 nmol per mouse). B. Images from control WT mice on normal chow. Fluorescence represented is from the aortic arch ROI only, with other non-heart region sources of fluorescence excluded for clarity. No fluorescence thresholding was applied to the tomographic datasets. Images show that there is a range of signal within the upper heart region ROI in ApoE-/- mice, but signal is much lower in wild-type (WT) control C57BL/6 mice.



Application notes and posters

  • Application note: Non-Invasive Quantitative In Vivo Imaging of Atherosclerosis Disease Progression and Treatment Response in ApoE Deficient Mice using Fluorescence Molecular Tomography and NIR Fluorescent Pre-clinical Imaging Agents


Citations

View our Citations Library to browse or search citations for in vivo imaging and atherosclerosis.