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Cavitation of microbubble contrast agents with ultrasound produces shear-mediated vasodilation and an increase in tissue perfusion. We investigated the influence of the size of the cavitation volume by comparing flow augmentation produced by two-dimensional (2D) versus three-dimensional (3D) therapeutic ultrasound. We also hypothesized that cavitation could augment flow beyond the ultrasound field through release of vasodilators that are carried downstream.In 11 rhesus macaques, cavitation of intravenously administered lipid-shelled microbubbles was performed in the proximal forearm flexor muscles unilaterally for 10 min. Ultrasound cavitation (1.3 MHz, 1.5 MPa peak negative pressure) was performed with 2D or 3D transmission with beam elevations of 5 and 25 mm, respectively, and pulsing intervals (PIs) sufficient to allow complete postdestruction refill (5 and 12 sec for 2D and 3D, respectively). Contrast ultrasound perfusion imaging was performed before and after cavitation, using multiplane assessment within and beyond the cavitation field in 1.5-cm increments. Cavitation in the hindlimb of mice using 2D ultrasound at a PI of 1 or 5 sec was performed to examine microvascular flow changes from cavitation in only arteries versus the microcirculation.In primates, the degree of muscle flow augmentation in the center of the cavitation field was similar for 2D and 3D conditions (five- to sixfold increase for both, P .01 vs baseline). The spatial extent of flow augmentation was only modestly greater for 3D cavitation because of an increase in perfusion with 2D transmission that was detected outside of the cavitation field. In mice, cavitation in the microvascular compartment (PI 5 sec) produced the greatest degree of flow augmentation, yet cavitation in the arterial compartment (PI 1 sec) still produced a three- to fourfold increase in flow (P .001 vs control). The mechanism for flow augmentation beyond the cavitation zone was investigated by in vitro studies that demonstrated cavitation-related release of vasodilators, including adenosine triphosphate and nitric oxide, from erythrocytes and endothelial cells.Compared with 2D transmission, 3D cavitation of microbubbles generates a similar degree of muscle flow augmentation, possibly because of a trade-off between volume of cavitation and PI, and only modestly increases the spatial extent of flow augmentation because of the ability of cavitation to produce conducted effects beyond the ultrasound field.

Background Platelet–endothelial interactions are thought to contribute to early atherogenesis. These interactions are potentiated by oxidative stress. We used in vivo molecular imaging to test the hypothesis that platelet–endothelial interactions occur at early stages of plaque development in obese, insulin‐resistant nonhuman primates, and are suppressed by NADPH‐oxidase‐2 inhibition. Methods and Results Six adult rhesus macaques fed a Western‐style diet for a median of 4.0 years were studied at baseline and after 8 weeks of therapy with the NADPH‐oxidase‐2‐inhibitor apocynin (50 mg/kg per day). Six lean control animals were also studied. Measurements included intravenous glucose tolerance test, body composition by dual‐energy X‐ray absorptiometry, carotid intimal medial thickness, carotid artery contrast ultrasound molecular imaging for platelet GPIbα (glycoprotein‐ Ibα) and vascular cell adhesion molecule‐1, and blood oxidative markers on mass spectrometry. Compared with lean controls, animals on a Western‐style diet were obese (median body mass: 16.0 versus 8.7 kg, P =0.003; median truncal fat: 49% versus 20%, P =0.002), were insulin resistant (4‐fold higher insulin–glucose area under the curve on intravenous glucose tolerance test, P =0.002), had 40% larger carotid intimal medial thickness ( P =0.004), and exhibited oxidative signatures on proteomics. In obese but not lean animals, signal enhancement on molecular imaging was significantly elevated for GPIbα and vascular cell adhesion molecule‐1. The signal correlated modestly with intimal medial thickness but not with the degree of insulin resistance. Apocynin significantly ( P 80% and vascular cell adhesion molecule‐1 signal by 75%, but did not affect intimal medial thickness, body mass, or intravenous glucose tolerance test results. Conclusion In nonhuman primates, diet‐induced obesity and insulin resistance leads to platelet–endothelial adhesion at early atherosclerotic lesion sites, which is associated with the expression of pro‐inflammatory adhesion molecules. These responses appear to be mediated, in part, through oxidative pathways.

BACKGROUND: Ultrasound-mediated cavitation of microbubble contrast agents produces high intravascular shear. We hypothesized that microbubble cavitation increases myocardial microvascular perfusion through shear-dependent purinergic pathways downstream from ATP release that is immediate and sustained through cellular ATP channels such as Pannexin-1. METHODS: Quantitative myocardial contrast echocardiography (MCE) perfusion imaging and in vivo optical imaging of ATP was performed in wild-type and Pannexin-1-deficient (Panx1(−/−)) mice before, 5 and 30 minutes after 10 minutes of ultrasound-mediated (1.3 MHz, mechanical index 1.3) myocardial microbubble cavitation. Flow augmentation in a pre-clinical model closer to humans was evaluated in rhesus macaques undergoing MCE perfusion imaging after high-power cavitation in the apical 4-chamber plane for 10 minutes. RESULTS: Microbubble cavitation in wild-type mice (n=7) increased myocardial perfusion by 64±25% at 5 minutes and 95±55% at 30 minutes compared to baseline (p

Echocardiographic molecular imaging techniques are beginning to be applied to evaluate preclinical efficacy of new drugs. In a large clinical trial, anti-interleukin-1β (IL-1β) immunotherapy reduced atherosclerotic events, yet treatment effects were modest, and the mechanisms of action were not fully elucidated. We tested the hypothesis that echocardiographic molecular imaging can assess changes in vascular thromboinflammatory status in response to anti-IL-1β therapy.In wild-type and atherosclerotic mice deficient for the low-density lipoprotein-receptor and Apobec-1, closed-chest myocardial infarction (MI) was performed to mimic high-risk clinical cohorts. Control animals had sham surgery. Post-MI animals were randomized to either no therapy or anti-IL-1β immunotherapy, which was continued weekly. At post-MI day 3 or 21, in vivo ultrasound molecular imaging of aortic VCAM-1, P-selectin, von Willebrand factor A1-domain, and platelet GPIbα in the thoracic aorta was performed. Aortic histology and NF-κB activity were assessed in atherosclerotic mice.In both atherosclerotic and wild-type mice, MI produced a several-fold increase (P .05) in aortic molecular signals for P-selectin, VCAM-1, von Willebrand factor, and GPIbα. In atherosclerotic mice, signal remained elevated at day 21. Anti-IL-1β therapy completely abolished the post-MI increase in signal for all endothelial targets (P .05 vs nontreated) at day 3 and 21. In atherosclerotic mice, MI triggered an increase in aortic plaque growth and macrophage content, a decrease in plaque collagen, and elevated aortic NF-κB (P .05 for all changes). All of these remote plaque adverse changes were inhibited by anti-IL-1β therapy.Echocardiographic molecular imaging of the vascular endothelium can quantify the beneficial effects of therapies designed to suppress the proatherosclerotic arterial thromboinflammatory effects of alarmins such as IL-1β. This approach could potentially be used to evaluate the biologic variables that influence response in preclinical studies, and possibly to select patients most likely to benefit from therapy.

The third-generation tyrosine kinase inhibitor (TKI) ponatinib has been associated with high rates of acute ischemic events. The pathophysiology responsible for these events is unknown. We hypothesized that ponatinib produces an endothelial angiopathy involving excessive endothelial-associated von Willebrand factor (VWF) and secondary platelet adhesion. In wild-type mice and ApoE−/− mice on a Western diet, ultrasound molecular imaging of the thoracic aorta for VWF A1-domain and glycoprotein-Ibα was performed to quantify endothelial-associated VWF and platelet adhesion. After treatment of wild-type mice for 7 days, aortic molecular signal for endothelial-associated VWF and platelet adhesion were five- to sixfold higher in ponatinib vs sham therapy (P < .001), whereas dasatinib had no effect. In ApoE−/− mice, aortic VWF and platelet signals were two- to fourfold higher for ponatinib-treated compared with sham-treated mice (P < .05) and were significantly higher than in treated wild-type mice (P < .05). Platelet and VWF signals in ponatinib-treated mice were significantly reduced by N-acetylcysteine and completely eliminated by recombinant ADAMTS13. Ponatinib produced segmental left ventricular wall motion abnormalities in 33% of wild-type and 45% of ApoE−/− mice and corresponding patchy perfusion defects, yet coronary arteries were normal on angiography. Instead, a global microvascular angiopathy was detected by immunohistochemistry and by intravital microscopy observation of platelet aggregates and nets associated with endothelial cells and leukocytes. Our findings reveal a new form of vascular toxicity for the TKI ponatinib that involves VWF-mediated platelet adhesion and a secondary microvascular angiopathy that produces ischemic wall motion abnormalities. These processes can be mitigated by interventions known to reduce VWF multimer size.

Background: Platelet interactions with the vascular endothelium contribute to early atherogenesis, and occur in part from oxidative stress and secondary dysregulation of endothelial-associated von Willebrand factor (VWF). We used in vivo contrast enhanced ultrasound (CEU) molecular imaging of the carotid endothelium in a non-human primate model of diet-induced obesity to test whether platelet adhesion is present in atherosclerosis-susceptible arteries prior to plaque development, and whether it can be modified through NADPH-oxidase (Nox) inhibition. Methods: Six adult rhesus macaques fed a high-fat diet (HFD), with 30% of calories from fat, for >2 years were studied at baseline and after 8 weeks of therapy with the Nox inhibitor apocynin (50 mg/kg/d). Six lean chow-fed controls were also studied. Intravenous glucose tolerance tests (IVGTT) and body composition were assessed at each interval. CEU molecular imaging of VCAM-1 and platelet GPIbα of the carotid arteries bilaterally was used to assess endothelial activation and platelet adhesion. Carotid intimal thickening (IMT) and brachial flow-mediated dilation (FMD) were assessed by ultrasound. Results: Before therapy, animals on HFD compared to controls were obese (16.0 vs 9.3 kg, p=0.003), had increased visceral adiposity (49% vs 25% truncal fat, p=0.002), and were insulin resistant (4-fold higher insulin AUC on IVGTT, p=0.002). FMD and IMT were similar between cohorts, although mild plaque was seen in the carotid bulb in 3 HFD macaques. HFD animals had greater (p2 =0.50, p=0.0001). Apocynin significantly reduced (p Conclusions: Platelet- endothelial interactions occur early in atherosclerosis-prone carotid arteries of obese and insulin resistant non-human primates, and correlates with the degree of endothelial adhesion molecule expression. Inhibition of Nox suppresses platelet adhesion and reduces VCAM-1 expression independent of any effects on insulin resistance and obesity.

In the months after acute myocardial infarction (MI), risk for acute atherothrombotic events in nonculprit arteries increases several fold.This study investigated whether sustained proinflammatory and prothrombotic endothelial alterations occur in remote vessels after MI.Wild-type mice, atherosclerotic mice with double knockout (DKO) of the low-density lipoprotein receptor and Apobec-1, and DKO mice treated with the Nox-inhibitor apocynin were studied at baseline and at 3 and 21 days after closed-chest MI. Ultrasound molecular imaging of P-selectin, vascular cell adhesion molecule (VCAM)-1, von Willebrand factor (VWF) A1-domain, and platelet GPIbα was performed. Intravital microscopy was used to characterize post-MI leukocyte and platelet recruitment in the remote microcirculation after MI.Aortic molecular imaging for P-selectin, VCAM-1, VWF-A1, and platelets was increased several-fold (p 0.01) 3 days post-MI for both wild-type and DKO mice. At 21 days, these changes resolved in wild-type mice but persisted in DKO mice. Signal for platelet adhesion was abolished 1 h after administration of ADAMTS13, which regulates VWF multimerization. In DKO and wild-type mice, apocynin significantly attenuated the post-MI increase for molecular targets, and platelet depletion significantly reduced P-selectin and VCAM-1 signal. On intravital microscopy, MI resulted in remote vessel leukocyte adhesion and platelet string or net complexes. On histology, high-risk inflammatory features in aortic plaque increased in DKO mice 21 days post-MI, which were completely prevented by apocynin.Acute MI stimulates a spectrum of changes in remote vessels, including up-regulation of endothelial inflammatory adhesion molecules and platelet-endothelial adhesion from endothelial-associated VWF multimers. These remote arterial alterations persist longer in the presence of hyperlipidemia, are associated with accelerated plaque growth and inflammation, and are attenuated by Nox inhibition.

This study evaluated whether lipoprotein apheresis produces immediate changes in resting perfusion in subjects with severe hypercholesterolemia, and whether there is a difference in the response between peripheral and coronary microcirculations.Lipoprotein apheresis is used in patients with severe hypercholesterolemia to reduce plasma levels of low-density lipoprotein cholesterol.Quantitative contrast-enhanced ultrasound perfusion imaging of the myocardium at rest and skeletal muscle at rest and during calibrated contractile exercise was performed before and immediately after lipoprotein apheresis in 8 subjects with severe hypercholesterolemia, 7 of whom had a diagnosis of familial hypercholesterolemia. Myocardial perfusion imaging was also performed in 14 normal control subjects. Changes in myocardial work and left ventricular function were assessed by echocardiography. Ex vivo ovine coronary and femoral artery ring tension assays were assessed in the presence of pre- and post-apheresis plasma.Apheresis acutely decreased low-density lipoprotein cholesterol (234.9 ± 103.2 mg/dl vs. 67.1 ± 49.5 mg/dl; p 0.01) and oxidized phospholipid on apolipoprotein B-100 (60.2 ± 55.2 nmol/l vs. 47.0 ± 24.5 nmol/l; p = 0.01), and acutely increased resting myocardial perfusion (55.1 [95% confidence interval: 77.2 to 73.1] IU/s vs. 135 [95% confidence interval: 81.2 to 189.6] IU/s; p = 0.01), without changes in myocardial work. Myocardial longitudinal strain improved in those subjects with reduced pre-apheresis function. Skeletal muscle perfusion at rest and during contractile exercise was unchanged by apheresis. Acetylcholine-mediated dilation of ex vivo ovine coronary but not femoral arteries was impaired in pre-apheresis plasma and was completely reversed in post-apheresis plasma.Lipoprotein apheresis produces an immediate improvement in coronary microvascular function, which increases myocardial perfusion and normalizes endothelial-dependent vasodilation. These changes are not observed in the periphery. (Acute Microvascular Changes With LDL Apheresis; NCT02388633).

The rise of diabetes in Kenya presents many challenges to a healthcare system struggling to deal with communicable diseases such as HIV and malaria. The lack of a proper system in place to quickly and effectively diagnose diabetes, especially in remote areas, has led to many preventable complications and a rapid increase in diabetes-related hospital admissions. Based on a review of literature and governmental reports, as well as fieldwork conducted in Nyeri, Kenya, this article describes patient screening and diagnosis pathways. The diverse human factors, technology challenges and systemic issues that impede the timely and widespread screening of diabetics are described in details. An understanding of the patient landscape and specific failure modes can inform technological, business and policy innovations to lower barriers to diabetes screening, diagnosis and management.