Contrast-enhanced Ultrasound Molecular Imaging Research Articles

Factors Affecting the Endothelial Retention of Targeted Microbubbles: Influence of Microbubble Shell Design and Cell Surface Projection of the Endothelial Target Molecule

In biologic systems, the arrest of circulating cells is mediated by adhesion molecules projecting their active binding domain above the cell surface to enhance bond formation and tether strength. Similarly, molecular spacers are used for ligands on particle-based molecular imaging agents. The aim of this study was to evaluate the influence of tether length for targeting ligands on ultrasound molecular imaging agents. Microbubbles bearing biotin at the end of variable-length polyethylene glycol spacer arms (MB(2000) and MB(3400)) were prepared. To assess invivo attachment efficiency to endothelial counterligands that vary in their distance from the endothelial cell surface, contrast-enhanced ultrasound (CEU) molecular imaging of tumor necrosis factor-α-induced P-selectin (long distance) or intercellular adhesion molecule-2 (short distance) was performed with each agent in murine hind limbs. To assess the influence of the glycocalyx on microbubble attachment, CEU molecular imaging of intercellular adhesion molecule-2 was performed after degradation of the glycocalyx. CEU molecular imaging targeted to P-selectin showed signal enhancement above control agent for MB(2000) and MB(3400), the degree of which was significantly higher for MB(3400) compared with MB(2000). CEU molecular imaging targeted to intercellular adhesion molecule-2 showed low overall signal for all agents and signal enhancement above control for MB(3400) only. Glycocalyx degradation increased signal for MB(3400) and MB(2000). Microbubble targeting to endothelial ligands is influenced by (1) the tether length of the ligand, (2)the degree to which the endothelial target is projected from the cell surface, and (3) the status of the glycocalyx. These considerations are important for designing targeted imaging probes and understanding potential obstacles to molecular imaging.

Oral Abstract Sessions: Young Investigator Award session * Thursday 8 December 2011, 12:45-13:45 * Location: Pecs

Oral Abstract Sessions: Young Investigator Award session * Thursday 8 December 2011, 12:45-13:45 * Location: Pecs

Abstract 9926: Molecular Imaging of the Pro-angiogenic Paracrine Effects of Stem Cell Therapy

Background: Pro-angiogenic stem cells are a promising therapy for patients with severe coronary or peripheral vascular disease. Mechanisms by which cell therapy promotes vascular remodeling remain unclear. We applied contrast-enhanced ultrasound (CEU) molecular imaging to test whether cell therapy acts in a paracrine fashion by promoting recruitment of pro-angiogenic (CX3CR-1 hi ) monocytes. Methods: Unilateral hindlimb ischemia was produced in 52 mice by iliac artery ligation. One day later, the proximal hindlimb received either: (1) IM injection of 1×10 6 multipotent adult progenitor cells (MAPC), (2) sham saline injection, or (3) no therapy. At Day3, 7, and 21 CEU perfusion imaging and molecular imaging of CX3CR-1 and P-selectin were performed. In 8 separate mice, intravital microscopy of TNF-a-treated cremaster muscle was performed 3 days after injection of either 5×10 5 MAPCs or saline. Results: Arterial ligation resulted in a 70% reduction in limb muscle blood flow to 0.20±0.06 ml/min/g. MAPC therapy produced a more rapid and complete recovery of flow (0.61±0.06 ml/min/g at day 21, ≈25% higher than both control groups, p<0.05). Signal for CX3CR-1 was significantly (p<0.05) higher vs control groups at all 3 study intervals (Figure),corroborated by a marked increase in tissue monocytes on Mac-2 immunohistology. P-selectin signal was also significantly higher in the MAPC-treated mice at days 3 and 7 (Figure). Intravital microscopy which showed that MAPCs migrated to a perivascular location and produced a 50% increase in leukocyte rolling flux fraction. Conclusions: In chronic limb ischemia, MAPCs migrate to a perivascular location and promote the sustained recruitment of pro-angiogenic monocytes, in part through effects on endothelial adhesion molecule expression. These results suggest that cell therapy can act to promote vascular remodeling through inflammatory pathways

Contrast Ultrasound and Targeted Microbubbles: Diagnostic and Therapeutic Applications for Angiogenesis

Angiogenesis represents the formation of new capillaries from existing vasculature, and as such plays a critical role in the response to ischemia in the setting of chronic coronary artery and peripheral vascular disease. Recent technological advances in non-invasive imaging modalities now allow the molecular imaging of angiogenesis. One such technique is contrast-enhanced ultrasound using microbubbles targeted against molecular markers of the angiogenic process. The ability to non-invasively image the angiogenic process would be useful in risk stratifying patients with arterial occlusive disease and would aid in the evaluation of new therapies to promote angiogenesis in ischemic cardiac and skeletal muscle. Furthermore, ultrasound technologies have also been developed that allow targeted angiogenic gene therapy using high-power ultrasound and DNA-bearing microbubbles. This review will focus specifically on recent advances in (1) contrast-enhanced ultrasound molecular imaging techniques for the evaluation of angiogenesis and (2) ultrasound-mediated gene delivery for therapeutic angiogenesis, techniques that have potential for translation to clinical practice.

Molecular imaging using contrast-enhanced ultrasound: evaluation of angiogenesis and cell therapy

The field of regenerative medicine and its applications for cardiovascular diseases continues to grow rapidly, fuelled by the increasing numbers of symptomatic patients who are not candidates for conventional revascularization procedures and remain refractory to maximal medical therapy. Therapeutic angiogenesis, initially in the form of the administration of growth factor protein or gene therapy and, more recently, in the form of adult progenitor cell therapy, has emerged as a promising new method of treatment for patients with ischaemic heart disease and peripheral arterial disease. There is a growing interest in non-invasive imaging techniques to evaluate the response to angiogenic gene-and cell-based therapies. Contrast-enhanced ultrasound (CEU) techniques using site-specific microbubbles have recently been developed for the molecular imaging of the vascular phenotype that characterizes angiogenesis. These methods have now been modified to allow the imaging of progenitor cell engraftment into neovessels. These molecular imaging techniques using contrast ultrasound and targeted microbubbles have the potential to further characterize the angiogenic response, aid in the optimization of gene- and cell-based strategies of therapeutic neovascularization, and ultimately serve to monitor the therapeutic effects in patients enrolled in clinical trials of regenerative therapies. This review will focus specifically on CEU molecular imaging techniques for the evaluation of angiogenesis and cell therapies in cardiovascular diseases, including: (i) an overview of the techniques and results of pre-clinical studies; (ii) a comparison of CEU molecular imaging techniques with other available molecular imaging modalities; and (iii) a discussion of the future role of CEU molecular imaging in the field of regenerative medicine.

Molecular Imaging of Inflammation in Atherosclerosis With Targeted Ultrasound Detection of Vascular Cell Adhesion Molecule-1

The ability to image vascular inflammatory responses may allow early diagnosis and treatment of atherosclerosis. We hypothesized that molecular imaging of vascular cell adhesion molecule-1 (VCAM-1) expression with contrast-enhanced ultrasound (CEU) could be used for this purpose. Attachment of VCAM-1-targeted and control microbubbles to cultured endothelial cells was assessed in a flow chamber at variable shear stress (0.5 to 12.0 dynes/cm2). Microbubble attachment to aortic plaque was determined by en face microscopy of the thoracic aorta 10 minutes after intravenous injection in wild-type or apolipoprotein E-deficient mice on either chow or hypercholesterolemic diet. CEU molecular imaging of the thoracic aorta 10 minutes after intravenous microbubble injection was performed for the same animal groups. VCAM-1-targeted but not control microbubbles attached to cultured endothelial cells, although firm attachment at the highest shear rates (8 to 12 dynes/cm2) occurred only in pulsatile flow conditions. Aortic attachment of microbubbles and targeted CEU signal was very low in control wild-type mice on chow diet. Aortic attachment of microbubbles and CEU signal for VCAM-1-targeted microbubbles differed between treatment groups according to extent of VCAM-1-positive plaque formation (median CEU videointensity, 1.8 [95% CI, 1.2 to 1.7], 3.7 [95% CI, 2.9 to 7.3], 6.8 [95% CI, 3.9 to 7.6], and 11.2 [95% CI, 8.5 to 16.0] for wild-type mice on chow and hypercholesterolemic diet and for apolipoprotein E-deficient mice on chow and hypercholesterolemic diet, respectively; P<0.001). CEU molecular imaging of VCAM-1 is capable of rapidly quantifying vascular inflammatory changes that occur in different stages of atherosclerosis. This method may be potentially useful for early risk stratification according to inflammatory phenotype.