The origins of contrast enhanced ultrasound imaging (CEUS) date to the earliest observations of Claude Joyner and publications of Gramiak and Shah in 1968.1 Interest in the development of ultrasound contrast agents and associated clinical applications continues today, nearly 40 years after the first reports.

The use of blood pool agents for enhancement of cardiovascular structures is ubiquitous, current clinical diagnostic imaging modalities utilize contrast agents to define anatomy and quantify tissue perfusion. Specifically, with regard to ultrasound contrast agents, the unique physical properties of air-filled, microspheres serving as true intravascular indicators provide an unparalleled access to the intrinsic spatial and temporal heterogeneity of tissue perfusion.2

Importantly, though initially developed for diagnostic applications, novel therapeutic applications for the ultrasound contrast agents are forthcoming. Thus, while the use of ultrasound contrast agents today provides important clinical information regarding chamber enhancement and myocardial perfusion, in the near future these agents will provide therapeutic options for site-specific, drug/gene delivery. Ultimately, ultrasound contrast agents may provide the opportunity to dramatically alter both the diagnosis and subsequent treatment of numerous diseases.

During the formative years of CEUS development, researchers focused on the development of novel agents and the requisite validation of the concept that these air-filled, microspheres represented true, intravascular, non-diffusible indicators. The list of scientists/clinicians performing this pioneering work in the 1960-1980’s include many of the following names: Gramiak and Shah,1 Reale et al,3 Meltzer et al,4 DeMaria et al,5,6 Reisner, Schwartz, Kremkau,7 Ziskin et al,8 Bove et al,9 Bommer et al,10 McKay and coworkers,11 Kort and Kronzon,12 Goldberg13 to list but a few of the innovators. The scientific investigations provided a foundation for the ensuing clinical applications of CEUS.

Later, a second wave of CEUS development was spearheaded by the efforts of Armstrong et al,14 Tei et al,15 Feinstein et al,16 Powsner et al,17 Kaul et al,18 Porter et al,19 Kemper et al,20 Zwehl et al,21 to list only a few. Of note, the initial efforts designed to quantify the novel “contrast effect” can be attributed to DeMaria and Bommer,6 Ong et al,22 Meltzer et al.23

Ultimately, the efforts culminated in the production of commercial ultrasound contrast agents (Levovist in Europe, Berlex, Schering, and Albunex in the USA, Molecular Biosystems Inc.).

Today, implementation of sophisticated harmonic imaging systems and lower mechanical index imaging provide prolonged in vivo persistence and markedly enhanced, signal to noise ratios. Importantly, the appropriate and clinically indicated clinical uses of CEUS are reimbursed through third party insurers based on the proven safety and efficacy of use.

The development of “second” generation contrast agents generally utilized high molecular weight, low soluble gases, and resulted in the prolonged in vivo persistence. These second generation agents fulfilled the required clinical expectations for safe, efficient, and economical non-invasive imaging of the left-sided cardiac chambers (i.e., left ventricular opacification, Doppler enhancement, and myocardial perfusion).

The “third” generation of contrast agents may be considered as “designer” agents for molecular imaging. These agents are uniquely and specifically labeled and, as such, are designed to provide quantitative, physiologic localization (“molecular imaging”) of inflammation, and related disease states. The leaders in this field include Jonathan Lindner, Lisa Villanueva, Thomas Porter, Evan Unger, Samuel Wickline, David McPherson, to name but a few.

Ultimately, the “fourth generation” CEUS applications break new ground in the area of therapy. These agents are designed as ultrasound-directed, site-specific drug/gene therapeutic systems. The pioneers in this field include Ishihara,24 Unger et al,25 Grayburn and coworkers,26 and an ever-expanding cadre of researchers.

Reflecting back to the early years of CEUS, it is clear that the dedicated work of many clinicians/researchers cannot be underestimated. Their research efforts directly contributed to the development of a novel, safe, and efficacious non-invasive imaging modality which improves patient care and reduces downstream testing expenses and risk.

Today, the clinical vascular applications for the use of CEUS are legion. Similar to the chamber enhancement applications for echocardiography, the vascular applications include enhancement of aorta, carotid arteries, and peripheral venous systems.

Mattrey and Kono initially identified the clinical value of using CEUS as an alternative to more invasive imaging technologies.35,36 Although, ultrasound contrast agents are not currently FDA approved in the USA, this is not the case in Europe, Asia, and South America. In the autumn of 2009, it is expected that vascular FDA-approved, clinical trials will be initiated in the USA.

Recently the use of CEUS has been identified as a novel imaging system capable of producing high resolution, real-time, images of microvascular perfusion, including tumor angiogenesis. Specifically, imaging of the neovasculature (vasa vasorum) within the carotid artery atherosclerotic plaque has captured world-wide attention.37,38,39 Thus, the unifying concept is that arterial wall inflammation/hypoxia provides a source for the generation of VEGF proteins and subsequent neovascularization growth. Hypoxia and inflammation events are routinely observed in diverse disease states including diabetes, atherosclerosis, connective tissue diseases, and cancer. And with great prescience, Judah Folkman realized that neovascularization provides the requisite tumor nutrient blood supply commonly observed in a variety of disease states.40

Ultrasound contrast agents serve as blood pool agents and consequently provide enhancement of the carotid arterial luminal surface which includes the common, bifurcation, and internal carotid arteries. Often the case, the ability to clearly define extent of the carotid vascular tree eludes the examiner. The image quality is often compromised by the physical habitus of the subject. Importantly, CEUS provides a non-ionizing radiation alternative to more invasive and higher risk procedures.36

Over the last few years, there are numerous reports from international centers which described the use of CEUS for vascular imaging, specifically for the anatomic enhancement of the luminal structures leading to the diagnosis of irregular surface lesions, ulcers, soft plaque, etc.2,35,37,41-45

At Rush University Medical Center in Chicago, Illinois, we have performed over 1000 CEUS clinical carotid examinations since 2001. All the studies were performed under physician supervision with appropriate institutional approvals. No adverse events or untoward occurrences were noted. Remarkably, over the last 9 years, frequently, luminal irregularities (i.e., ulcers and “soft plaque”) were not observed without the use of ultrasound contrast agents (see Figures 1, 2, 3, and 4). In order to confirm the value of CEUS for routine vascular imaging, multi-center, prospective clinical trials will be required to ascertain the value of using these agents for enhanced lesion detection in “at risk” populations.37

Figure 1 Contrast-enhanced carotid ultrasound imaging—intra-luminal plaque. A, C Two different carotid arteries with intra-luminal plaque on B-mode ultrasound imaging. B, D Corresponding arteries on contrast-enhanced ultrasound. The carotid intima-media complex (c-IMT) is depicted as a hypoechoic line and the adventitial layer appears echogenic

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Figure 2 Contrast-enhanced carotid ultrasound imaging—plaque ulceration. A, C Two different carotid arteries with plaque ulceration on B-mode ultrasound imaging. B, D Corresponding arteries on contrast-enhanced ultrasound

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Figure 3 Contrast-enhanced carotid ultrasound imaging—intraplaque neovascularization. Carotid artery with intra-luminal plaques and intra-plaque neovascularization (arrow)

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Figure 4 Contrast-enhanced, vascular imaging. The image on the left revealed the un-enhanced, carotid ultrasound image. The images on the right revealed contrast-enhanced, vascular imaging. The circled region in red, highlights the intra-luminal plaque with associated intra-plaque angiogenesis (microspheres appear as white objects within the plaque)

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The use of the ultrasound-derived c-IMT as a surrogate marker of systemic atherosclerosis was first described by Pignoli in 1986.46 Subsequently, over the last 23 years, the use of c-IMT has become a widely accepted clinical standard for the detection of premature atherosclerosis. Numerous FDA approved clinical trials utilized c-IMT as an efficient marker for therapeutic efficacy.47

Traditionally, clinicians and researchers readily acknowledge the technical limitations which surround the precise measurement of c-IMT measurements, particularly associated with identification of the carotid artery near wall IMT.48-50 The primary technical issues revolve around acoustic physics and include the reverberant acoustic noise generated from the overlying soft tissues; all of which limit a clear acoustic definition of the near wall. In distinction to the near wall, the acoustic definition of the carotid artery far wall is regularly and routinely visualized. This is due, in part, to the overlying presence of the uniform media (blood) and lack of tissue reflectance and interference. Due to the fact that the carotid artery far wall remains an acoustically strong acoustic reflector, this target is generally used in most clinical studies and related pharmaceutical trials.

However, based on recently published data, CEUS provides a reliable and precise measurement of the near c-IMT, particularly when compared to similar c-IMT measurements performed without the use of ultrasound contrast agents.51,52,45 In fact, and wholly consistent with this data, previous pathology studies indicate that the near wall is consistently thicker (20%) than the far wall of the c-IMT; thus, implying that systemic atherosclerosis is preferentially distributed on the near wall structures.53

Generally, the procedure for performing CEUS is routine and straightforward. Specifically, after a patient is referred for a clinically indicated carotid ultrasound examination, it is reasonable to develop a consent form for “off-label” use of ultrasound contrast agents. The performance of a CEUS study requires a peripheral intravenous injection of 0.5-1.0 mL of an FDA approved contrast agent. Standard vascular imaging of the carotid artery is routinely performed with a linear array transducer. The mechanical index used for CEUS is set at levels that are considerably lower levels than those required for non-contrast studies (0.1-0.2 MI vs 0.3-0.5 MI, respectively). In order to measure the c-IMT, it is recommended that one uses a readily available semi-automated, computer-assisted c-IMT software program.

The clinical utilization of the c-IMT as a surrogate marker for the diagnosis of premature atherosclerosis is increasing based on published clinical data bases. Accordingly, the American Society and the Society for Vascular Medicine have provided guidelines for clinical use.54

Clearly, a current limitation for the use of performing serial c-IMT measurements in individuals remains the inherent error of measurement due to the lack of a truly volumetric ultrasound scanner. While the value if two-dimensional c-IMT analyses is unquestioned, the issues surrounding the routine clinical use remain. Ultimately, with the introduction of real-time, three-dimensional ultrasound scanners, the technical issues associated with image alignment and registration will be reduced.

Historically, clinicians and researchers have reported upon the association of plaque vascularity and “vulnerability.”39,55-74 Recently, CEUS imaging of the carotid artery provided a novel, non-invasive method for directly examining plaque vascularity; perhaps providing a “window” into plaque vulnerability.

Historical consideration of the vasa vasorum

Atherosclerotic plaques are believed to develop from an initial endothelial cell insult often precipitated by mechanical shearing, oxidative, and/or hypoxia stresses from noxious substances. Following the initiating event, the subsequent deposition of intra-cellular matter promotes focal migration of inflammatory cells (monocyte derived macrophages), smooth muscle cells, and fibroblasts, accumulating in the intracellular space often resulting in foam cell development, raised lesions and release of tissue hypoxic factors, and VEGF proteins.

Sluimer et al, provided insight into the mechanism surrounding the ongoing development of the tissue hypoxia based on a description of the incomplete endothelial junctions and inadequate structural integrity of the immature and thin-walled microvessels.72 The presence of the immature microvessels (“leaky” vessels) contribute inflammation materials by providing a source of noxious plasma components (hemoglobin, oxidized low-density lipoprotein cholesterol, lipoprotein[a], glucose, advanced glycation end products AGE) and inflammatory cells.73,74

Ultimately, the atherosclerotic plaque, similar to the other abnormal tumor growths, requires nutrient blood flow supplied by arterial and venous vasa vasorum.

The anatomic structure of the vasa vasorum as related to the growth of atherosclerotic plaques has been well characterized by pathologists over 100 years ago.65

Based on a series of autopsy reports, intra-plaque angiogenic vessels were identified within the vessel wall (media and intima) in subjects with known systemic atherosclerosis.65,75 The seminal articles of Barger et al55 and Beeuwkes et al56 and Winternitz in 1876, provided important evidence directly linking adventitial and intra-plaque vasa vasorum to the atherosclerotic disease processes.

In 2004, Fleiner et al75 observed that the presence and degree of neovascularization within vulnerable plaque was associated with plaque rupture and clinical occlusive cardiovascular events.

Similarly, Kumamoto et al in 1995 observed:

There was a significant positive correlation between the density of new vessels in the intima and the incidence of luminal stenosis, the extent of chronic inflammatory infiltrate, the formation of granulation tissue, or the atheromatous changes, whereas the vascular density decreased in the extensively hyalinized and calcified intima. The newly formed intimal vessels originated mainly from the adventitial vasa vasorum and also partly from the proper coronary lumen. The intimal vessels that originated from the adventitia occurred approximately 28 times more frequently than those that originated from the luminal side.65

In addition, Moreno and Fuster reported findings which directly linked atherosclerosis and diabetes to the formation of vulnerable plaques.76 Recently, Mauriello et al examined 544 coronary segments in 16 patients who experienced fatal coronary events.77 The results revealed the presence of diffuse, active inflammation in the entire coronary vascular system, in patients with both stable and vulnerable plaques.

Using experimental animal models with dietary-induced atherosclerosis, Williams et al showed regression of intima and media neovascularization after a reduction of cholesterol feeding.78 In addition, Wilson et al79 used micro-computed tomography techniques to observe the induction of coronary adventitial vasa vasorum in the pig and, subsequently, revealed regression after initiating statin therapy. Importantly, the authors noted that the excessive growth of adventitial vasa vasorum preceded the development of luminal plaques. Similarly, Moulton et al studied anti-angiogenesis therapies in an experimental animal model of atherosclerosis.80

In 2007, Shah et al, at Rush University Medical Center, in Chicago, published a clinical validation study based on pathology specimens of subjects who underwent CEUS prior to undergoing carotid endarterectomy surgery.81 The results revealed a direct, positive correlation between CEUS images and the surgically derived, tissue specimens with regard to presence and degree of angiogenesis within the human carotid plaques. For this study, the histology consisted of hematoxylin and eosin stains, with immunohistochemical markers: CD31, CD34, von Willebrand factor, CD68, and were evaluated for degree of vascularity.

Sets of stained slides were examined microscopically for evidence of neovascularization and inflammation using a grading system as reported by Jeziorska in 1999.61

Similar to the limitation mentioned in performing serial c-IMT measurements in the individual patient, the ability to accurately quantify adventitial and intra-plaque vasa vasorum, will necessitate the construction of an ultrasound system with true, volumetric, image acquisition. Figure 5 is an image obtained from a real-time, 3D mechanical ultrasound system during a clinical study using CEUS. As noted, one can identify the luminal surfaces and the presence of the adventitial vasa vasorum.

Figure 5 Contrast-enhanced, 3D vascular imaging. The lumen of the carotid artery is opacified following the intravenous injection of an ultrasound contrast agent (white). Note that the luminal-intima interface is highlighted. The adventitial vasa vasorum are highlighted at the level of carotid bulb (white arrows)

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