2D echocardiography can be used to evaluate wall thickening, which is closely dependent on resting myocardial blood flow (MBF). Because myocardial contractility is a major determinant of myocardial oxygen consumption, reductions in resting MBF are followed within seconds by the development of a wall thickening abnormality.24) Furthermore, ischemic myocardial segments with low resting flow will demonstrate resting perfusion defects. Thus, myocardial contrast echocardiography (MCE) can determine whether patients with acute chest pain are suffering from an acute coronary syndrome (ACS). In a prospective study of 114 patients presenting to the emergency room with "suspected cardiac chest pain", myocardial perfusion defects demonstrated 77% sensitivity for the detection of ACS compared to 28% and 34% respectively with ECG and troponin while maintaining similar specificity (89-96%).25) Abnormal myocardial perfusion was the only independent variable for diagnosing an ACS (odds ratio = 87, p < 0.001).

The short and long-term prognostic significance of MCE has also been shown in chest pain patients.26) Patients with abnormal perfusion were 2.5-fold more likely to have non-fatal myocardial infarction or cardiac death, but those with both abnormal regional function and myocardial perfusion were 14.3-fold (p < 0.001) more likely to have events - demonstrating the incremental benefit of combined wall thickening and perfusion data over regional function alone in these patients.

The diagnostic and predictive value of contrast echocardiography in acute chest pain is limited by a number of factors, however. When a patient has only ischemia but no infarction, wall thickening abnormalities (stunning) resolves over time - so perfusion and function may both have returned to normal if a patient presents late after their insult. In patients with prior infarction, it may be difficult to determine if a wall thickening abnormality is due to acute ischemia, or to remote infarction. In such cases, targeted imaging to identify recent ischemia-reperfusion injury (ischemic memory imaging) may be very valuable.

In one study, Ley et al.26) chose to detect the presence of P-selectin upregulation after ischemia. P-selectin is an endothelial adhesion molecule which is transported to the endothelial cell surface rapidly after an inflammatory stimulus, where it participates in leukocyte capture and rolling on the venular surface.26) The presence of P-selectin can persist for many hours after the initial injury. The anterior myocardium of mice were subjected to 10 min of ischemia followed by 45 min of reperfusion to allow recovery of resting function. Biotinylated microbubbles conjugated with a monoclonal antibody targeted against P-selectin were administered after reperfusion and showed selective retention and contrast enhancement of the post-ischemic anterior wall at a time when both myocardial perfusion and wall thickening had normalized.27) In Fig. 4, P-selectin targeted microbubbles were administered in open-chest dogs which had been subjected to 90 min of ischemia followed by reperfusion. The area of contrast enhancement has been color-coded so that green to yellow to red reflect greater signal intensity. Panels A and B demonstrate separate animals with left anterior descending (Fig. 4A), and left circumflex (Fig. 4B) territory ischemia followed by 60 min of reperfusion. Intense retention of targeted microbubbles resulting in bright enhancement is seen in the reperfused beds of the respective coronary arteries.28) Yan et al.29) evaluated the ability of molecular ultrasound to detect the presence of ICAM-1, which is upregulated on the venular endothelial surface later than P-selectin, but persists for much longer - with peak expression at 24 h after reperfusion. Accordingly, biotinylated microbubbles conjugated with a monoclonal antibody targeted against ICAM-1 were administered at 1, 8 and 24 h after a brief 15 min ischemic insult in mice. Selective enhancement of the post-ischemic anterior myocardium was demonstrated at the 8 and 24 h time points using targeted microbubbles. At 1 h, though, anti-ICAM-1 targeted microbubbles did not cause greater enhancement than microbubbles conjugated with a control antibody.29) These experiments demonstrate the dramatic ability of targeted imaging to identify myocardium which has been ischemic even up to 24 h after the insult. The endothelial target of choice will vary depending on the interval between the ischemic insult and the time of imaging (P-selectin early and ICAM-1 late), which highlights the need for a user to have good understanding of molecular biology to take full advantage of this technology.

Another condition characterized by inflammation is acute heart transplant rejection. Rats that had undergone heterotopic cardiac transplantation with and without acute rejection were administered microbubbles targeted to ICAM-1. Persistent diffuse myocardial contrast enhancement was noted in rejecting allografts after administration of targeted microbubbles, while no enhancement was seen after administration of control microbubbles.30)

Atheromatous lesions are sites of endothelial dysfunction, intimal thickening, and there is overexpression of adhesion molecules to mediate leukocyte infiltration into plaque.31) The ability to detect early atherosclerosis in vivo, prior to the development of a significantly stenotic lesion or a cardiovascular event, could potentially be used to identify high risk individuals in need of aggressive treatment and risk-factor modification for primary prevention. The ability of microbubbles labeled with vascular cell adhesion molecule-1 (VCAM-1) to identify early atherosclerotic lesions in the aorta of apo-E knockout mouse has now been demonstrated.32) The degree of contrast enhancement was related to the extent of plaque, so targeted microbubbles can not only detect the presence of early atheromatous lesions, but also quantify the degree of associated inflammation.32)

The development of new blood vessels can be due to remodeling of new or pre-existing coronary collaterals (arteriogenesis), to the formation of new blood vessels from endothelial progenitor cells (vasculogenesis), or to the formation of new blood vessels from pre-existing vessels (angiogenesis). Contrast ultrasound could play 2 roles in angiogenesis - targeted microbubbles could be used to monitor the presence and development of new blood vessels, and microbubbles could also be used to deliver drugs or genes to promote and maintain an angiogenic response. This latter role will be discussed further below.

Imaging angiogenesis has utilized agents that have been targeted against integrins (such as α_v -integrins like α_vβ₃ or α_vβ₅), against growth factors or their receptors (e.g. vascular endothelial growth factor or VEGF2 receptors), and against endothelial cell markers (such as VCAM-1).

The angiogenic models that have been used for imaging include tumor neovessels, matrigel plugs impregnated with fibroblast growth factor-2 (FGF-2) to stimulate angiogenesis development from surrounding vessels, and ischemia models. For example, microbubbles bearing antibodies to VEGF2 receptor have been shown to enhance murine breast cancer models,33) contrast agents conjugated to small peptides which have strong affinity integrins like the arginine-glycine-aspartate (RGD) containing disintegrin echistatin have been used to enhance human squamous cell carcinoma implanted in nude mice,34) or arginine-arginine-leucine peptide has been used to detect Clone C and PC3 tumors in mice.35) The microbubbles selectively adhere to tumor-derived rather normal endothelium, which suggests that targeted contrast ultrasound has the potential to be used to characterize tumor angiogenesis, and subsequently to monitor antitumor or antiangiogenic therapies.

Fig. 5 shows the ability of targeted agents to detect neovessels in a murine model using a Matrigel plug impregnated with FGF-2 which stimulates angiogenesis from surrounding vessels. The microbubbles were conjugated with a monoclonal antibody against α_v - integrins and produced significant enhancement of angiogenesis that had developed around the periphery of the plug (Fig. 5B).36) Similar success was shown using microbubbles conjugated to RGD peptide containing disintegrin echistatin (Fig. 5C).36)37) Conversely, no signal enhancement was noted when imaging was performed using a non-specific isotype antibody (Fig. 5A).36)

Targeted microbubbles and ischemia models have been used to evaluate the role of angiogenesis in improving perfusion to ischemic tissue, and have also been used to assess the role of growth factors on enhancing angiogenesis. Using a chronic hind-limb ischemia model in the rat of unilateral iliac artery ligation, the natural arteriogenic response to ischemia was demonstrated using a microbubble conjugated to a RGD-peptide, and the augmentation in neovascularization that occurred when FGF-2 was administered to the ischemic hindlimb was shown.38) Behm et al.39) then used microbubbles targeted to neutrophils, monocyte α₅ - integrins, and VCAM-1 at different time points after iliac ligation. They demonstrated that early after ligation, all 3 components were present, followed by a precipitous decline in neutrophil signal after 2-4 days after ligation, with persistence of monocyte and VCAM signal until day 7. Improvement in tissue perfusion increased much more slowly, not peaking until day 21 post-ligation.39) This study demonstrated the power of targeted contrast ultrasound to evaluate separate factors which contribute to angiogenesis both functionally and temporally.

Gene therapy is limited by lack of a safe and effective method for gene delivery. Viral vectors have potential for immunogenic and cytotoxic effects. Plasmid delivery is safe, but have low transfection rates even with direct injection.

Microbubbles can potentially be used for therapeutic purposes, by enhancing the delivery of genes or drugs to specific targets. Microbubble destruction appears to be an important aspect of this method, especially when the microbubbles are destroyed in close proximity to the endothelial cell surface such as when the microbubbles attached to endothelial cells, or lodged in small arterioles.40) The mechanism by which microbubbles enhance gene/drug uptake is not entirely clear, but it is possible that temporary cellular membrane poration can be produced by shell fragmentation, which could enhance the uptake of DNA into perivascular cells.40-42) Shell implantation could also be enhanced through the generation of high-velocity pressure jets, heat or free radicals which are produced in the vicinity of the endothelial surface by microbubble destruction.

Microbubbles bearing viral vectors, plasmid DNA and antisense oligonucleotides have been used to enhance delivery to tissues. Fig. 5 shows extravascular deposits of plasmid containing luciferase cDNA which was charge-coupled to the surface of cationic microbubbles under fluorescent microscopy.40) The fluorescent DNA can be seen in the perivascular muscle adjacent to microvessels. These experiments demonstrated that no DNA deposition occurred in the absence of ultrasound. Furthermore, there was significantly greater DNA deposition following intra-arterial administration of microbubbles compared to intravenous, because the microbubbles with the former would not be subjected to pulmonary filtering and would become lodged within very small arterioles and capillaries. Despite the fact that microbubbles were being destroyed in close proximity to the endothelial surface, the vast majority of deposition events (85-90%) occurred without visible vascular ruptures or hemorrhage.40) The most efficient deposition occurred with intra-arterial injection, and with high power ultrasound exposure. If either component was missing, there was much less transfection (Fig. 6).