Brian P. Davidson

Molecular Imaging of Inflammation and Platelet Adhesion in Advanced Atherosclerosis Effects of Antioxidant Therapy With NADPH Oxidase Inhibition

Background—In atherosclerosis, local generation of reactive oxygen species amplifies the inflammatory response and contributes to plaque vulnerability. We used molecular imaging to test whether inhibition of NADPH oxidase with apocynin would reduce endothelial inflammatory activation and endothelial–platelet interactions, thereby interrupting progression to high-risk plaque phenotype. Methods and Results—Mice deficient for both the low-density lipoprotein receptor and Apobec-1 were studied at 30 weeks of age and again after 10 weeks with or without apocynin treatment (10 or 50 mg/kg per day orally). In vivo molecular imaging of vascular cell adhesion molecule-1 (VCAM 1) P-selectin, and platelet glycoprotein-1b&agr; (GPIb&agr;) in the thoracic aorta was performed with targeted contrast-enhanced ultrasound molecular imaging. Arterial elastic modulus and pulse wave transit time were assessed using ultrahigh frequency ultrasound and invasive hemodynamic measurements. Plaque size and composition were assessed by histology. Molecular imaging in nontreated mice detected a 2-fold increase in P-selectin expression, VCAM-1 expression, and platelet adhesion between 30 and 40 weeks of age. Apocynin reduced all of these endothelial events in a dose-dependent fashion (25% and 50% reduction in signal at 40 weeks for low- and high-dose apocynin). Apocynin also decreased aortic elastic modulus and increased the pulse transit time. On histology, apocynin reduced total monocyte accumulation in a dose-dependent manner as well as platelet adhesion, although total plaque area was reduced in only the high-dose apocynin treatment group. Conclusions—Inhibition of NADPH oxidase in advanced atherosclerosis reduces endothelial activation and platelet adhesion, which are likely responsible for the arrest of plaque growth and improvement of vascular mechanical properties.

Ultrasound-Mediated Vascular Gene Transfection by Cavitation of Endothelial-Targeted Cationic Microbubbles

OBJECTIVES Ultrasound-mediated gene delivery can be amplified by acoustic disruption of microbubble carriers that undergo cavitation. We hypothesized that endothelial targeting of microbubbles bearing cDNA is feasible and, through optimizing proximity to the vessel wall, increases the efficacy of gene transfection. BACKGROUND Contrast ultrasound-mediated gene delivery is a promising approach for site-specific gene therapy, although there are concerns with the reproducibility of this technique and the safety when using high-power ultrasound. METHODS Cationic lipid-shelled decafluorobutane microbubbles bearing a targeting moiety were prepared and compared with nontargeted microbubbles. Microbubble targeting efficiency to endothelial adhesion molecules (P-selectin or intercellular adhesion molecule [ICAM]-1) was tested using in vitro flow chamber studies, intravital microscopy of tumor necrosis factor-alpha (TNF-α)-stimulated murine cremaster muscle, and targeted contrast ultrasound imaging of P-selectin in a model of murine limb ischemia. Ultrasound-mediated transfection of luciferase reporter plasmid charge coupled to microbubbles in the post-ischemic hindlimb muscle was assessed by in vivo optical imaging. RESULTS Charge coupling of cDNA to the microbubble surface was not influenced by the presence of targeting ligand, and did not alter the cavitation properties of cationic microbubbles. In flow chamber studies, surface conjugation of cDNA did not affect attachment of targeted microbubbles at microvascular shear stresses (0.6 and 1.5 dyne/cm(2)). Attachment in vivo was also not affected by cDNA according to intravital microscopy observations of venular adhesion of ICAM-1-targeted microbubbles and by ultrasound molecular imaging of P-selectin-targeted microbubbles in the post-ischemic hindlimb in mice. Transfection at the site of high acoustic pressures (1.0 and 1.8 MPa) was similar for control and P-selectin-targeted microbubbles but was associated with vascular rupture and hemorrhage. At 0.6 MPa, there were no adverse bioeffects, and transfection was 5-fold greater with P-selectin-targeted microbubbles. CONCLUSIONS We conclude that ultrasound-mediated transfection at safe acoustic pressures can be markedly augmented by endothelial juxtaposition.

Molecular Imaging of the Paracrine Proangiogenic Effects of Progenitor Cell Therapy in Limb Ischemia

Background— Stem cells are thought to enhance vascular remodeling in ischemic tissue in part through paracrine effects. Using molecular imaging, we tested the hypothesis that treatment of limb ischemia with multipotential adult progenitor cells (MAPCs) promotes recovery of blood flow through the recruitment of proangiogenic monocytes. Methods and Results— Hind-limb ischemia was produced in mice by iliac artery ligation, and MAPCs were administered intramuscularly on day 1. Optical imaging of luciferase-transfected MAPCs indicated that cells survived for 1 week. Contrast-enhanced ultrasound on days 3, 7, and 21 showed a more complete recovery of blood flow and greater expansion of microvascular blood volume in MAPC-treated mice than in controls. Fluorescent microangiography demonstrated more complete distribution of flow to microvascular units in MAPC-treated mice. On ultrasound molecular imaging, expression of endothelial P-selectin and intravascular recruitment of CX3CR-1-positive monocytes were significantly higher in MAPC-treated mice than in the control groups at days 3 and 7 after arterial ligation. Muscle immunohistology showed a >10-fold-greater infiltration of monocytes in MAPC-treated than control-treated ischemic limbs at all time points. Intravital microscopy of ischemic or tumor necrosis factor-&agr;–treated cremaster muscle demonstrated that MAPCs migrate to perimicrovascular locations and potentiate selectin-dependent leukocyte rolling. In vitro migration of human CD14+ monocytes was 10-fold greater in response to MAPC-conditioned than basal media. Conclusions— In limb ischemia, MAPCs stimulate the recruitment of proangiogenic monocytes through endothelial activation and enhanced chemotaxis. These responses are sustained beyond the MAPC lifespan, suggesting that paracrine effects promote flow recovery by rebalancing the immune response toward a more regenerative phenotype.

Detection of Antecedent Myocardial Ischemia With Multiselectin Molecular Imaging

OBJECTIVES Our aim was to develop an echocardiographic molecular imaging approach for detecting recent myocardial ischemia by using recombinant P-selectin glycoprotein ligand (PSGL)-1 as a targeting ligand, which is a feasible approach for human use. BACKGROUND Ischemic memory imaging using human PSGL-1 as a targeting moiety may extend the time window for postischemic detection by targeting the early (P-selectin) and late (E-selectin) endothelial ischemic response. METHODS Lipid microbubbles bearing recombinant human PSGL-1 (MB(YSPSL)) or P-selectin antibody (MB(Ab)) were prepared. Targeted attachment was evaluated by using flow chamber and intravital microscopy. In vivo ultrasound molecular imaging was first performed in the hindlimb in wild-type and P-selectin-deficient (P(-/-)) mice 45 to 360 min after brief ischemia-reperfusion injury. Myocardial contrast echocardiography molecular imaging was performed 1.5, 3, 6, and 18 h after brief left anterior descending coronary artery ischemia-reperfusion. RESULTS Microbubble attachment to P-selectin-immunoglobulin G fusion protein in flow chamber experiments (shear stress 0.5 to 8.0 dyne/cm(2)) and to activated venular endothelium on intravital microscopy were similar for MB(Ab) and MB(YSPSL). Intense enhancement was seen for MB(Ab) and MB(YSPSL) in postischemic muscle and was more stable over time for MB(YSPSL). On myocardial contrast echocardiography, both MB(YSPSL) and MB(Ab) produced similar signal enhancement at 90 min and 3 h after ischemia, which spatially correlated with the postischemic risk area. Signal significantly decreased but was still present at 6 to 18 h. CONCLUSIONS Echocardiographic molecular imaging with a human multi-selectin-targeted contrast agent bearing recombinant human PSGL-1 can detect myocardial ischemia hours after resolution. This approach may potentially be used for rapid bedside evaluation of patients with recent chest pain.

Dysregulated Selectin Expression and Monocyte Recruitment During Ischemia-Related Vascular Remodeling in Diabetes Mellitus

Objective—Diabetes mellitus (DM) is associated with impaired ischemia-related vascular remodeling and also dysregulation of the inflammatory response. We sought to determine whether impaired selectin-mediated monocyte recruitment in ischemic tissues contributes to blunted ischemia-mediated angiogenesis in DM. Methods and Results—Contrast-enhanced ultrasound perfusion imaging and molecular imaging of endothelial P-selectin expression in the proximal hindlimb were performed at 1, 3, and 21 days after arterial ligation in wild-type and db/db mice. Ligation reduced muscle blood flow to ≈0.05 mL/minute per gram in both strains. Significant recovery of flow occurred only in wild-type mice (60%–65% of baseline flow). On molecular imaging, baseline P-selectin signal was 4-fold higher in db/db compared with wild-type mice (P<0.01) but increased minimally at day 1 after ischemia, whereas signal increased approximately 10-fold in wild-type mice (P<0.01). Immunohistology of the hindlimb skeletal muscle demonstrated severely reduced monocyte recruitment in db/db mice compared with wild-type mice. Local treatment with monocyte chemotactic protein-1 corrected the deficits in postischemic P-selectin expression and monocyte recruitment in db/db mice and led to greater recovery in blood flow. Conclusion—In DM, there is dysregulation of the selectin response to limb ischemia, which leads to impaired monocyte recruitment, which may be mechanistically related to reduced vascular remodeling in limb ischemia.

Molecular Imaging of Platelet–Endothelial Interactions and Endothelial von Willebrand Factor in Early and Mid-Stage Atherosclerosis

Background—Nonthrombotic platelet–endothelial interactions may contribute to atherosclerotic plaque development, although in vivo studies examining mechanism without platelet preactivation are lacking. Using in vivo molecular imaging at various stages of atherosclerosis, we quantified platelet–endothelial interactions and evaluated the contribution of major adhesion pathways. Methods and Results—Mice deficient for the low-density lipoprotein receptor and Apobec-1 were studied as an age-dependent model of atherosclerosis at 10, 20, 30, and 40 weeks of age, which provided progressive increase in stage from early fatty streak (10 weeks) to large complex plaques without rupture (40 weeks). Platelet-targeted contrast ultrasound molecular imaging of the thoracic aorta performed with microbubbles targeted to GPIb&agr; demonstrated selective signal enhancement as early as 10 weeks of age. This signal increased progressively with age (almost 8-fold increase from 10 to 40 weeks, analysis of variance P<0.001). Specificity for platelet targeting was confirmed by the reduction in platelet-targeted signal commensurate with the decrease in platelet count after immunodepletion with anti-GPIb or anti-CD41 antibody. Inhibition of P-selectin in 20 and 40 weeks atherosclerotic mice resulted in a small (15% to 30%) reduction in platelet signal. Molecular imaging with microbubbles targeted to the A1 domain of von Willebrand factor demonstrated selective signal enhancement at all time points, which did not significantly increase with age. Treatment of 20 and 40 week mice with recombinant ADAMTS13 eliminated platelet and von Willebrand factor molecular imaging signal. Conclusions—Platelet–endothelial interactions occur in early atherosclerosis. These interactions are in part caused by endothelial von Willebrand factor large multimers, which can be reversed with exogenous ADAMTS13.

Temporal Characterization of the Functional Density of the Vasa Vasorum by Contrast-Enhanced Ultrasonography Maximum Intensity Projection Imaging

OBJECTIVES We sought to determine whether contrast-enhanced ultrasound (CEU) microangiography with maximum intensity projection (MIP) processing could temporally evaluate proliferation of the vasa vasorum (VV) in a model of mural hemorrhage. BACKGROUND Expansion of the VV and plaque neovascularization contributes to plaque growth and instability and may be triggered by a variety of stimuli, including vascular hemorrhage. However, quantitative in vivo methods for temporal assessment of VV remodeling are lacking. METHODS In 24 rabbits fed a high-fat diet, either autologous whole blood or saline was percutaneously injected into the media-adventitia of the femoral artery using ultrahigh-frequency ultrasound guidance. Functional VV density at the injection site and contralateral control artery was assessed 1, 2, and 6 weeks after injection with CEU imaging with MIP processing. In vitro studies with renathane microtubes were also performed to validate linear density measurement with CEU and MIP processing. RESULTS In vitro studies demonstrated that MIP processing of CEU data reflected the relative linear density of vessels in a manner that was relatively independent of contrast concentration or microtube flow rate. On CEU with MIP, there was a 3-fold increase in femoral artery VV microvascular density at 1 and 2 weeks after blood injection (p < 0.01 vs. contralateral control), whereas VV density increased minimally after saline injection. At 6 weeks, VV vascular density decreased in blood-treated vessels and was not different from saline-injected or contralateral control vessels. CONCLUSIONS CEU with MIP processing can provide quantitative data on temporal changes in the functional density of the VV. This method may be useful for evaluating high-risk features of plaque neovascularization or response to therapies aimed at plaque neovessels.

Ischemic memory imaging in nonhuman primates with echocardiographic molecular imaging of selectin expression.

BACKGROUND Selectins are adhesion molecules that are expressed by the vascular endothelium upon activation and may be an imaging target for detecting myocardial ischemia long after resolution. The aim of this study was to test the hypothesis that molecular imaging of selectins with myocardial contrast echocardiographic (MCE) molecular imaging could be used to detect recent brief ischemia in closed-chest nonhuman primates. METHODS Myocardial ischemia was produced in anesthetized adult rhesus macaques (n = 6) by percutaneous balloon catheter occlusion of the left anterior descending or circumflex coronary artery for 5 to 10 min. Three separate macaques served as nonischemic controls. MCE perfusion imaging was performed during coronary occlusion to measure risk area and at 100 to 110 min to exclude infarction. MCE molecular imaging was performed at 30 and 90 min after reperfusion using a lipid microbubble bearing dimeric recombinant human P-selectin glycoprotein ligand-1 (MB-YSPSL). Collection of blood for safety data, electrocardiography, and echocardiography were performed at baseline and before and 10 min after each MB-YSPSL injection. RESULTS Vital signs, oxygen saturation, electrocardiographic results, ventricular systolic function, pulmonary vascular resistance, and serum safety markers were unchanged by intravenous injection of MB-YSPSL. On echocardiography, left ventricular dysfunction in the risk area had resolved by 30 min, and there was no evidence of infarction on MCE perfusion imaging. On selectin-targeted MCE molecular imaging, signal enhancement was greater (P < .05) in the risk area than remote territory at 30 min (25 ± 11 vs 11 ± 4 IU) and 90 min (13 ± 3 vs 3 ± 2 IU) after ischemia. There was no enhancement (<1 IU) in control nonischemic subjects. CONCLUSIONS In primates, MCE molecular imaging of selectins using MB-YSPSL, a recombinant ligand appropriate for humans, is both safe and effective for imaging recent myocardial ischemia. This technique may be useful for detecting recent ischemia in patients with chest pain even in the absence of necrosis.

Future applications of contrast echocardiography

Contrast echocardiography encompasses a broad variety of different applications whereby cardiovascular ultrasound is used to detect microbubble contrast agents that produce a strong acoustic signal. Ultrasound contrast agents are currently approved for use in cardiology for the purposes of opacifying the left ventricular chamber in order to aid interpretation of echocardiography, yet their off-label use has been applied for myocardial perfusion imaging in ischaemic heart disease for over a decade. This article provides a broad overview of some of the future uses of contrast ultrasound in patients with cardiovascular disease that are on the horizon, including: (1) evaluating vascular anatomy and plaque neovascularisation; (2) detecting abnormal microvascular function; (3) assessing peripheral vascular disease with stress/rest limb perfusion imaging; (4) molecular imaging of cardiovascular disease; (5) site targeted delivery of therapeutic genes or drugs; and (6) ultrasound facilitated thrombolysis. Signal enhancement during contrast enhanced ultrasound (CEU) relies on the detection of the acoustic energy produced by encapsulated microbubbles or other acoustically active particles. The basis for signal generation from these contrast agents is their compressibility.w1 w2 Ultrasound contrast agents contain gases or, less commonly, emulsions with a low vapour pressure. Hence, they are several orders of magnitude more compressible than water or tissue and, because they are also smaller than the wavelength of diagnostic ultrasound, undergo volumetric oscillation in the pressure fluctuations of an ultrasound field.w1 The ‘vibration’ of microbubbles produces, among other forms of energy (light, heat), strong backscattered acoustic pressure signals that produce contrast enhancement on ultrasound imaging. As predicted by the Rayleigh–Plesset principles, the magnitude of bubble oscillation and, hence, signal generation is influenced by the compressibility and density of the gas, the frequency and power of …

CONTRAST-ENHANCED ULTRASOUND ASSESSMENT OF IMPAIRED ADIPOSE TISSUE AND MUSCLE PERFUSION IN INSULIN-RESISTANT MICE

Background—In diabetes mellitus, reduced perfusion and capillary surface area in skeletal muscle, which is a major glucose storage site, contribute to abnormal glucose homeostasis. Using contrast-enhanced ultrasound, we investigated whether abdominal adipose tissue perfusion is abnormal in insulin resistance and correlates with glycemic control. Methods and Results—Contrast-enhanced ultrasound perfusion imaging of abdominal adipose tissue and skeletal muscle was performed in obese insulin resistance (db/db) mice at 11 to 12 or 14 to 16 weeks of age and in control lean mice. Time–intensity data were analyzed to quantify microvascular blood flow (MBF) and capillary blood volume (CBV). Blood glucose response for 1 hour was measured after insulin challenge (1 U/kg, IP). Compared with control mice, db/db mice at 11 to 12 and 14 to 16 weeks had a higher glucose concentration area under the curve after insulin (11.8±2.8, 20.6±4.3, and 28.4±5.9 mg·min/dL [×1000], respectively; P=0.0002) and also had lower adipose MBF (0.094±0.038, 0.035±0.010, and 0.023±0.01 mL/min per gram; P=0.0002) and CBV (1.6±0.6, 1.0±0.3, and 0.5±0.1 mL/100 g; P=0.0017). The glucose area under the curve correlated in a nonlinear fashion with both adipose and skeletal muscle MBF and CBV. There were significant linear correlations between adipose and muscle MBF (r=0.81) and CBV (r=0.66). Adipocyte cell volume on histology was 25-fold higher in 14- to 16-week db/db versus control mice. Conclusions—Abnormal adipose MBF and CBV in insulin resistance can be detected by contrast-enhanced ultrasound and correlates with the degree of impairment in glucose storage. Abnormalities in adipose tissue and muscle seem to be coupled. Impaired adipose tissue perfusion is in part explained by an increase in adipocyte size without proportional vascular response.