J. Todd Belcik

Molecular Imaging of the Initial Inflammatory Response in Atherosclerosis Implications for Early Detection of Disease

Background—We hypothesized that molecular imaging of endothelial cell adhesion molecule expression could noninvasively evaluate prelesion atherogenic phenotype. Methods and Results—Mice deficient for the LDL-receptor and the Apobec-1 editing peptide (DKO mice) were studied as an age-dependent model of atherosclerosis. At 10, 20, and 40 weeks of age, ultrasound molecular imaging of the proximal thoracic aorta was performed with contrast agents targeted to P-selectin and VCAM-1. Atherosclerotic lesion severity and content were assessed by ultrahigh frequency ultrasound, histology, and immunohistochemistry. In wild-type mice at all ages, there was neither aortic thickening nor targeted tracer signal enhancement. In DKO mice, lesions progressed from sparse mild intimal thickening at 10 weeks to widespread severe lesions with luminal encroachment at 40 weeks. Molecular imaging for P-selectin and VCAM-1 demonstrated selective signal enhancement (P<0.01 versus nontargeted agent) at all ages for DKO mice. P-selectin and VCAM-1 signal in DKO mice were greater by 3-fold at 10 weeks, 4- to 6-fold at 20 weeks, and 9- to 10-fold at 40 weeks compared to wild-type mice. En face microscopy demonstrated preferential attachment of targeted microbubbles to regions of lesion formation. Conclusions—Noninvasive ultrasound molecular imaging of endothelial activation can detect lesion-prone vascular phenotype before the appearance of obstructive atherosclerotic lesions.

Guidelines for the Cardiac Sonographer in the Performance of Contrast Echocardiography: A Focused Update from the American Society of Echocardiography

Thomas R. Porter, MD, FASE (Chair), Sahar Abdelmoneim, MD, J. Todd Belcik, BS, RCS, RDCS, FASE, Marti L. McCulloch,MBA, RDCS, FASE, Sharon L.Mulvagh,MD, FASE, Joan J. Olson, BS, RDCS, RVT, FASE, Charlene Porcelli, BS, RDCS, RDMS, FASE, Jeane M. Tsutsui, MD, and Kevin Wei, MD, FASE, Omaha, Nebraska; Rochester, Minnesota; Portland, Oregon; Houston, Texas; Charleston, South Carolina; S~ ao Paulo, Brazil

Real-time measurement of renal blood flow in healthy subjects using contrast-enhanced ultrasound

Current methods for measuring renal blood flow (RBF) are time consuming and not widely available. Contrast-enhanced ultrasound (CEU) is a safe and noninvasive imaging technique suitable for assessment of tissue blood flow, which has been used clinically to assess myocardial blood flow. We tested the utility of CEU in monitoring changes in RBF in healthy volunteers. We utilized CEU to monitor the expected increase in RBF following a high protein meal in healthy adults. Renal cortical perfusion was assessed by CEU using low mechanical index (MI) power modulation Angio during continuous infusions of Definity. Following destruction of tissue microbubbles using ultrasound at a MI of 1.0, the rate of tissue replenishment with microbubbles and the plateau acoustic intensity (AI) were used to estimate the RBF velocity and cortical blood volume, respectively. Healthy adults (n = 19, mean age 26.6 yr) were enrolled. The A.beta parameter of CEU, representing mean RBF increased by 42.8%from a baseline of 17.05 +/- 6.23 to 23.60 +/- 6.76 dB/s 2 h after the ingestion of the high-protein meal (P = 0.002). Similarly, there was a 37.3%increase in the beta parameter, representing the geometric mean of blood velocity after the high protein meal (P < 0.001). The change in cortical blood volume was not significant (P = 0.89). Infusion time of Definity was 6.3 +/- 2.0 min. The ultrasound contrast agent was tolerated well with no serious adverse events. CEU is a fast, noninvasive, and practical imaging technique that may be useful for monitoring renal blood velocity, volume, and flow.

Proinflammatory Endothelial Activation Detected by Molecular Imaging in Obese Nonhuman Primates Coincides With Onset of Insulin Resistance and Progressively Increases With Duration of Insulin Resistance

Background— Inflammation and insulin resistance (IR) are associated processes that potentiate risk for cardiovascular disease in obesity. The temporal relation between IR and inflammation is not completely characterized. We hypothesized that endothelial cell adhesion molecule expression in large arteries is an early event that coincides with diet-induced obesity and IR in primates. Methods and Results— Ten adult male rhesus macaques were studied at baseline and every 4 to 6 months on a high-fat diet for 2 years. Truncal fat, carotid intima-media thickness, plasma inflammatory biomarkers, and carotid P-selectin and vascular cell adhesion molecule-1 expression by contrast-enhanced ultrasound molecular imaging were assessed. Intravenous glucose tolerance test was performed at baseline and at 4 and 18 months. A high-fat diet produced a rapid increase (P<0.01) in weight, truncal fat, and degree of IR indicated by the insulin area under the curve and glucose disappearance rate on intravenous glucose tolerance test, all of which worsened minimally thereafter. Molecular imaging detected a progressive increase in endothelial cell adhesion molecule expression over time (5- to 7-fold greater than control agent signal at 2 years; P<0.01). Changes in intima-media thickness were not detected until 2 years and, although there was a trend toward an increase in plasma markers of inflammation (monocyte chemotactic protein-1, C-reactive protein), the pattern of increase varied considerably over time. Conclusions— In primates with diet-induced obesity, endothelial inflammatory activation is an early event that occurs coincident with the development of IR and long before any measurable change in carotid intima-media thickness. Endothelial activation is related more to the duration rather than to the severity of IR and is not mirrored by changes in plasma biomarkers.

Prognostic Value of Dipyridamole Stress Myocardial Contrast Echocardiography: Comparison With Single Photon Emission Computed Tomography

BACKGROUND Dipyridamole stress myocardial contrast echocardiography (MCE) can be used to detect coronary artery disease (CAD). Because it measures myocardial blood flow velocity in addition to measuring myocardial blood volume, it was hypothesized that it should have greater prognostic utility than single photon-emission computed tomography (SPECT), which measures only myocardial blood volume. Because blood flow mismatch precedes wall thickening (WT) abnormalities during demand ischemia, it was also postulated that perfusion on MCE would be superior to WT abnormalities on echocardiography for this purpose. METHODS The incidence of nonfatal myocardial infarction and cardiac death was determined in 261 patients with known or suspected CAD over a mean follow-up period of 14 months who underwent simultaneous dipyridamole stress MCE and 99mTc-sestamibi SPECT. Comparisons of survival curves were conducted with stratified (and unstratified) log-rank tests. RESULTS Abnormal results on MCE were found to be the best predictor of an adverse outcome (odds ratio, 23; 95% confidence interval, 6-201; P<.0001) and provided incremental prognostic value over clinical variables (age>60 years, the presence of >or=3 cardiac risk factors, known peripheral vascular disease, prior myocardial infarction, and left ventricular systolic function), inducible WT abnormalities, and SPECT. Prognoses were worst in patients who had both abnormal results on MCE and inducible WT abnormalities and best in those who had neither. Patients with abnormal results on MCE but no inducible WT abnormalities had intermediate outcomes. CONCLUSION In patients with known or suspected CAD undergoing dipyridamole stress, MCE provides powerful prognostic information that is superior to clinical variables, electrocardiography, left ventricular systolic function, WT analysis, and SPECT. MCE may therefore serve as a method of choice for myocardial perfusion assessment in patients with known or suspected CAD. Larger studies are needed to confirm these findings.

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.

Cardiovascular and Systemic Microvascular Effects of Anti-Vascular Endothelial Growth Factor Therapy for Cancer

OBJECTIVES This study sought to evaluate the contribution of microvascular functional rarefaction and changes in vascular mechanical properties to the development of hypertension and secondary ventricular remodeling that occurs with anti-vascular endothelial growth factor (VEGF) therapy. BACKGROUND Hypertension is a common side effect of VEGF inhibitors used in cancer medicine. METHODS Mice were treated for 5 weeks with an anti-murine VEGF-A monoclonal antibody, antibody plus ramipril, or sham treatment. Microvascular blood flow (MBF) and blood volume (MBV) were quantified by contrast-enhanced ultrasound in skeletal muscle, left ventricle (LV), and kidney. Echocardiography and invasive hemodynamics were used to assess ventricular function, dimensions and vascular mechanical properties. RESULTS Ambulatory blood pressure increased gradually over the first 3 weeks of anti-VEGF therapy. Compared with controls, anti-VEGF-treated mice had similar aortic elastic modulus and histological appearance, but a marked increase in arterial elastance, indicating increased afterload, and elevated plasma angiotensin II. Increased afterload in treated mice led to concentric LV remodeling and reduced stroke volume without impaired LV contractility determined by LV peak change in pressure over time (dp/dt) and the end-systolic dimension-pressure relation. Anti-VEGF therapy did not alter MBF or MBV in skeletal muscle, myocardium, or kidney; but did produce cortical mesangial glomerulosclerosis. Ramipril therapy almost entirely prevented the adverse hemodynamic effects, increased afterload, and LV remodeling in anti-VEGF-treated mice. CONCLUSIONS Neither reduced functional microvascular density nor major alterations in arterial mechanical properties are primary causes of hypertension during anti-VEGF therapy. Inhibition of VEGF leads to an afterload mismatch state, increased angiotensin II, and LV remodeling, which are all ameliorated by angiotensin-converting enzyme inhibition.

Contrast-enhanced ultrasound reveals real-time spatial changes in vascular perfusion during early implantation in the macaque uterus.

OBJECTIVE To use contrast-enhanced ultrasound (CEU) to quantify blood flow in the macaque uterus during early pregnancy. DESIGN Prospective nonhuman primate study. SETTING Oregon National Primate Research Center. ANIMALS Naturally cycling female rhesus macaques (Macaca mulatta). INTERVENTION(S) Female macaques were mated on days 11-14 of the cycle. Females were then imaged by CEU and Doppler ultrasound once every 3 days from day 21 through day 39 of the fertile cycle. MAIN OUTCOME MEASURE(S) Visualization and quantification of uterine vascular perfusion. RESULT(S) CEU identified the primary placental disc and underlying vessels approximately 2 days earlier than Doppler ultrasound was able to observe endometrial thickening. CEU revealed spatial differences in vascular perfusion between the endometrium, myometrium, and endometrial-myometrial (junctional) zone. Myometrium displayed the highest rate of blood flow (>10 mL/min/g tissue). There was less blood flow in the endometrium and junctional zone (<3 mL/min/g). A brief fall in progesterone was observed during early implantation, which was correlated with reduced blood flow to all three uterine compartments, but did not reduce flow to the placenta. CONCLUSIONS CEU provides a sensitive, noninvasive method to assess vascular perfusion of the uterus during embryo implantation in macaques. We propose CEU as a new diagnostic tool to monitor vascular changes associated with early pregnancy in women.