Alkystis Phinikaridou

Bisphosphonate-anchored PEGylation and radiolabeling of superparamagnetic iron oxide: long-circulating nanoparticles for in vivo multimodal (T1 MRI-SPECT) imaging

The efficient delivery of nanomaterials to specific targets for in vivo biomedical imaging is hindered by rapid sequestration by the reticuloendothelial system (RES) and consequent short circulation times. To overcome these two problems, we have prepared a new stealth PEG polymer conjugate containing a terminal 1,1-bisphosphonate (BP) group for strong and stable binding to the surface of ultrasmall-superparamagnetic oxide nanomaterials (USPIOs). This polymer, PEG(5)-BP, can be used to exchange the hydrophobic surfactants commonly used in the synthesis of USPIOs very efficiently and at room temperature using a simple method in 1 h. The resulting nanoparticles, PEG(5)-BP-USPIOs are stable in water or saline for at least 7 months and display a near-zero ζ-potential at neutral pH. The longitudinal (r1) and transverse (r2) relaxivities were measured at a clinically relevant magnetic field (3 T), revealing a high r1 of 9.5 mM–1 s–1 and low r2/r1 ratio of 2.97, making these USPIOs attractive as T1-weighted MRI contrast agents at high magnetic fields. The strong T1-effect was demonstrated in vivo, revealing that PEG(5)-BP-USPIOs remain in the bloodstream and enhance its signal 6-fold, allowing the visualization of blood vessels and vascular organs with high spatial definition. Furthermore, the optimal relaxivity properties allow us to inject a dose 4 times lower than with other USPIOs. PEG(5)-BP-USPIOs can also be labeled using a radiolabeled-BP for visualization with single photon emission computed tomography (SPECT), and thus affording dual-modality contrast. The SPECT studies confirmed low RES uptake and long blood circulation times (t1/2 = 2.97 h). These results demonstrate the potential of PEG(5)-BP-USPIOs for the development of targeted multimodal imaging agents for molecular imaging.

Noninvasive Magnetic Resonance Imaging Evaluation of Endothelial Permeability in Murine Atherosclerosis Using an Albumin-Binding Contrast Agent

Background— Endothelial dysfunction promotes atherosclerosis and precedes acute cardiovascular events. We investigated whether in vivo magnetic resonance imaging with the use of an albumin-binding contrast agent, gadofosveset, could detect endothelial damage associated with atherosclerosis in apolipoprotein E–deficient (ApoE−/−) mice. Furthermore, we tested whether magnetic resonance imaging could noninvasively assess endothelial function by measuring the endothelial-dependent vasodilation in response to acetylcholine. Methods and Results— ApoE−/− mice were imaged at 4, 8, and 12 weeks after commencement of a high-fat diet. Statin-treated ApoE−/− mice were scanned after 12 weeks of a high-fat diet. Wild-type mice were imaged before and 48 hours after injection of Russells viper venom, an endothelial toxin. Delayed enhancement magnetic resonance imaging and T1 mapping of the brachiocephalic artery, 30 minutes after injection of gadofosveset, showed increased vessel wall enhancement and relaxation rate (R1) with progression of atherosclerosis in ApoE−/−(R1 [s−1]: R4 weeks 2.42±0.35, R8 weeks 3.45±0.54, R12 weeks 3.83±0.52) and Russells viper venom–injected wild-type mice (R1=4.57±0.86). Conversely, wild-type (R1=2.15±0.34) and statin-treated ApoE−/− (R1=3.0±0.65) mice showed less enhancement. Uptake of gadofosveset correlated with Evans blue staining, morphological changes of endothelial cells, and widening of the cell-cell junctions, suggesting that uptake occurs in regions of increased vascular permeability. Endothelial-dependent vasomotor responses showed vasoconstriction of the arteries of the ApoE−/− (−22.22±7.95%) and Russells viper venom–injected (−10.37±17.60%) mice compared with wild-type mice (32.45±12.35%). Statin treatment improved endothelium morphology and function (−8.12±8.22%). Conclusions— We demonstrate the noninvasive assessment of endothelial permeability and function with the use of an albumin-binding magnetic resonance contrast agent. Blood albumin leakage could be a surrogate marker for the in vivo evaluation of interventions that aim to restore the endothelium.

Detection of intracoronary thrombus by magnetic resonance imaging in patients with acute myocardial infarction.

Background— Persistent intracoronary thrombus after plaque rupture is associated with an increased risk of subsequent myocardial infarction and mortality. Coronary thrombus is usually visualized invasively by x-ray coronary angiography. Non–contrast-enhanced T1-weighted magnetic resonance (MR) imaging has been useful for direct imaging of carotid thrombus and intraplaque hemorrhage by taking advantage of the short T1 of methemoglobin present in acute thrombus and intraplaque hemorrhage. The aim of this study was to investigate the use of non–contrast–enhanced MR for direct thrombus imaging (MRDTI) in patients with acute myocardial infarction. Methods and Results— Eighteen patients (14 men; age, 61±9 years) underwent MRDTI within 24 to 72 hours of presenting with an acute coronary syndrome before invasive x-ray coronary angiography; MRDTI was performed with a T1-weighted, 3-dimensional, inversion-recovery black-blood gradient-echo sequence without contrast administration. Ten patients were found to have intracoronary thrombus on x-ray coronary angiography (left anterior descending, 4; left circumflex, 2; right coronary artery, 4; and right coronary artery–posterior descending artery, 1), and 8 had no visible thrombus. We found that MRDTI correctly identified thrombus in 9 of 10 patients (sensitivity, 91%; posterior descending artery thrombus not detected) and correctly classified the control group in 7 of 8 patients without thrombus formation (specificity, 88%). The contrast-to-noise ratio was significantly greater in coronary segments containing thrombus (n=10) compared with those without visible thrombus (n=131; mean contrast-to-noise ratio, 15.9 versus 2.6; P<0.001). Conclusion— Use of MRDTI allows selective visualization of coronary thrombus in a patient population with a high probability of intracoronary thrombosis.

Noninvasive MRI Evaluation of Endothelial Permeability in Murine Atherosclerosis Using an Albumin-Binding Contrast Agent

Background— Endothelial dysfunction promotes atherosclerosis and precedes acute cardiovascular events. We investigated whether in vivo magnetic resonance imaging with the use of an albumin-binding contrast agent, gadofosveset, could detect endothelial damage associated with atherosclerosis in apolipoprotein E–deficient (ApoE−/−) mice. Furthermore, we tested whether magnetic resonance imaging could noninvasively assess endothelial function by measuring the endothelial-dependent vasodilation in response to acetylcholine. Methods and Results— ApoE−/− mice were imaged at 4, 8, and 12 weeks after commencement of a high-fat diet. Statin-treated ApoE−/− mice were scanned after 12 weeks of a high-fat diet. Wild-type mice were imaged before and 48 hours after injection of Russells viper venom, an endothelial toxin. Delayed enhancement magnetic resonance imaging and T1 mapping of the brachiocephalic artery, 30 minutes after injection of gadofosveset, showed increased vessel wall enhancement and relaxation rate (R1) with progression of atherosclerosis in ApoE−/−(R1 [s−1]: R4 weeks 2.42±0.35, R8 weeks 3.45±0.54, R12 weeks 3.83±0.52) and Russells viper venom–injected wild-type mice (R1=4.57±0.86). Conversely, wild-type (R1=2.15±0.34) and statin-treated ApoE−/− (R1=3.0±0.65) mice showed less enhancement. Uptake of gadofosveset correlated with Evans blue staining, morphological changes of endothelial cells, and widening of the cell-cell junctions, suggesting that uptake occurs in regions of increased vascular permeability. Endothelial-dependent vasomotor responses showed vasoconstriction of the arteries of the ApoE−/− (−22.22±7.95%) and Russells viper venom–injected (−10.37±17.60%) mice compared with wild-type mice (32.45±12.35%). Statin treatment improved endothelium morphology and function (−8.12±8.22%). Conclusions— We demonstrate the noninvasive assessment of endothelial permeability and function with the use of an albumin-binding magnetic resonance contrast agent. Blood albumin leakage could be a surrogate marker for the in vivo evaluation of interventions that aim to restore the endothelium.

Porphyromonas gingivalis Accelerates Inflammatory Atherosclerosis in the Innominate Artery of ApoE Deficient Mice

OBJECTIVE Studies in humans support a role for the oral pathogen Porphyromonas gingivalis in the development of inflammatory atherosclerosis. The goal of this study was to determine if P. gingivalis infection accelerates inflammation and atherosclerosis in the innominate artery of mice, an artery which has been reported to exhibit many features of human atherosclerotic disease, including plaque rupture. METHODS AND RESULTS Apolipoprotein E-deficient (ApoE-/-) mice were orally infected with P. gingivalis, and magnetic resonance imaging (MRI) was used to monitor the progression of atherosclerosis in live mice. P. gingivalis infected mice exhibited a statistically significant increase in atherosclerotic plaque in the innominate artery as compared to uninfected mice. Polarized light microscopy and immunohistochemistry revealed that the innominate arteries of infected mice had increased lipids, macrophages and T cells as compared to uninfected mice. Increases in plaque, total cholesterol esters and cholesterol monohydrate crystals, macrophages, and T cells were prevented by immunization with heat-killed P. gingivalis prior to pathogen exposure. CONCLUSIONS These are the first studies to demonstrate progression of inflammatory plaque accumulation in the innominate arteries by in vivo MRI analysis following pathogen exposure, and to document protection from plaque progression in the innominate artery via immunization.

Role of miR-195 in aortic aneurysmal disease.

Rationale: Abdominal aortic aneurysms constitute a degenerative process in the aortic wall. Both the miR-29 and miR-15 families have been implicated in regulating the vascular extracellular matrix. Objective: Our aim was to assess the effect of the miR-15 family on aortic aneurysm development. Methods and Results: Among the miR-15 family members, miR-195 was differentially expressed in aortas of apolipoprotein E–deficient mice on angiotensin II infusion. Proteomics analysis of the secretome of murine aortic smooth muscle cells, after miR-195 manipulation, revealed that miR-195 targets a cadre of extracellular matrix proteins, including collagens, proteoglycans, elastin, and proteins associated with elastic microfibrils, albeit miR-29b showed a stronger effect, particularly in regulating collagens. Systemic and local administration of cholesterol-conjugated antagomiRs revealed better inhibition of miR-195 compared with miR-29b in the uninjured aorta. However, in apolipoprotein E–deficient mice receiving angiotensin II, silencing of miR-29b, but not miR-195, led to an attenuation of aortic dilation. Higher aortic elastin expression was accompanied by an increase of matrix metalloproteinases 2 and 9 in mice treated with antagomiR-195. In human plasma, an inverse correlation of miR-195 was observed with the presence of abdominal aortic aneurysms and aortic diameter. Conclusions: We provide the first evidence that miR-195 may contribute to the pathogenesis of aortic aneurysmal disease. Although inhibition of miR-29b proved more effective in preventing aneurysm formation in a preclinical model, miR-195 represents a potent regulator of the aortic extracellular matrix. Notably, plasma levels of miR-195 were reduced in patients with abdominal aortic aneurysms suggesting that microRNAs might serve as a noninvasive biomarker of abdominal aortic aneurysms.

In vivo detection of vulnerable atherosclerotic plaque by MRI in a rabbit model

Background—The ability to identify atherosclerotic plaques with a high risk for sudden disruption before stroke or myocardial infarction would be of great utility. We used a rabbit model of controlled atherothrombosis to test whether in vivo MRI can noninvasively distinguish between plaques that disrupt after pharmacological triggering (vulnerable) and those that do not (stable). Methods and Results—Atherosclerosis was induced in male New Zealand White (n=17) rabbits by cholesterol diet and endothelial denudation of the abdominal aorta. After baseline (pretrigger) MRI with and without gadolinium contrast, the rabbits underwent 2 pharmacological triggerings to induce atherothrombosis, followed by another MRI 48 hours later (post-triggering). Atherosclerosis was identified by the pretriggered images in all rabbits, and thrombosis was identified in 9 of 17 animals (53%) by post-trigger MRI. After the animals were euthanized, 95 plaques were analyzed; 28 (29.5%) had thrombi (vulnerable) and 67 did not (stable) (70.5%). Pretriggered MRI revealed comparable stenosis in stable and vulnerable plaques, but vulnerable plaques had a larger plaque area (4.8±1.6 versus 3.0±1.0 mm2; P=0.01), vessel area (9.2±3.0 versus. 15.8±4.9 mm2; P=0.01), and higher remodeling ratio (1.16±0.2 versus 0.93±0.2; P=0.01) compared with stable plaques. Furthermore, vulnerable plaques more frequently exhibited (1) positive remodeling (67.8% versus 22.3%; P=0.01), in which the plaque is hidden within the vessel wall instead of occluding the lumen; and (2) enhanced gadolinium uptake (78.6% versus 20.9%; P=0.01) associated with histological findings of neovascularization, inflammation, and tissue necrosis. Conclusions—We demonstrate that in vivo MRI at 3.0 T detects features of vulnerable plaques in an animal model of controlled atherothrombosis. These findings suggest that MRI may be used as a noninvasive modality for localization of plaques that are prone to disruption.

In vivo Detection of Vulnerable Atherosclerotic Plaque by Magnetic Resonance Imaging in a Rabbit Model

Background—The ability to identify atherosclerotic plaques with a high risk for sudden disruption before stroke or myocardial infarction would be of great utility. We used a rabbit model of controlled atherothrombosis to test whether in vivo MRI can noninvasively distinguish between plaques that disrupt after pharmacological triggering (vulnerable) and those that do not (stable). Methods and Results—Atherosclerosis was induced in male New Zealand White (n=17) rabbits by cholesterol diet and endothelial denudation of the abdominal aorta. After baseline (pretrigger) MRI with and without gadolinium contrast, the rabbits underwent 2 pharmacological triggerings to induce atherothrombosis, followed by another MRI 48 hours later (post-triggering). Atherosclerosis was identified by the pretriggered images in all rabbits, and thrombosis was identified in 9 of 17 animals (53%) by post-trigger MRI. After the animals were euthanized, 95 plaques were analyzed; 28 (29.5%) had thrombi (vulnerable) and 67 did not (stable) (70.5%). Pretriggered MRI revealed comparable stenosis in stable and vulnerable plaques, but vulnerable plaques had a larger plaque area (4.8±1.6 versus 3.0±1.0 mm2; P=0.01), vessel area (9.2±3.0 versus. 15.8±4.9 mm2; P=0.01), and higher remodeling ratio (1.16±0.2 versus 0.93±0.2; P=0.01) compared with stable plaques. Furthermore, vulnerable plaques more frequently exhibited (1) positive remodeling (67.8% versus 22.3%; P=0.01), in which the plaque is hidden within the vessel wall instead of occluding the lumen; and (2) enhanced gadolinium uptake (78.6% versus 20.9%; P=0.01) associated with histological findings of neovascularization, inflammation, and tissue necrosis. Conclusions—We demonstrate that in vivo MRI at 3.0 T detects features of vulnerable plaques in an animal model of controlled atherothrombosis. These findings suggest that MRI may be used as a noninvasive modality for localization of plaques that are prone to disruption.

Magnetic Resonance T1 Relaxation Time of Venous Thrombus Is Determined by Iron Processing and Predicts Susceptibility to Lysis

Background— The magnetic resonance longitudinal relaxation time (T1) changes with thrombus age in humans. In this study, we investigate the possible mechanisms that give rise to the T1 signal in venous thrombi and whether changes in T1 relaxation time are informative of the susceptibility to lysis. Methods and Results— Venous thrombosis was induced in the vena cava of BALB/C mice, and temporal changes in T1 relaxation time correlated with thrombus composition. The mean T1 relaxation time of thrombus was shortest at 7days following thrombus induction and returned to that of blood as the thrombus resolved. T1 relaxation time was related to thrombus methemoglobin formation and further processing. Studies in inducible nitric oxide synthase (iNOS−/−)–deficient mice revealed that inducible nitric oxide synthase mediates oxidation of erythrocyte lysis–derived iron to paramagnetic Fe3+, which causes thrombus T1 relaxation time shortening. Studies using chemokine receptor-2–deficient mice (Ccr2−/−) revealed that the return of the T1 signal to that of blood is regulated by removal of Fe3+ by macrophages that accumulate in the thrombus during its resolution. Quantification of T1 relaxation time was a good predictor of successful thrombolysis with a cutoff point of <747 ms having a sensitivity and specificity to predict successful lysis of 83% and 94%, respectively. Conclusions— The source of the T1 signal in the thrombus results from the oxidation of iron (released from the lysis of trapped erythrocytes in the thrombus) to its paramagnetic Fe3+ form. Quantification of T1 relaxation time appears to be a good predictor of the success of thrombolysis.

Regions of Low Endothelial Shear Stress Colocalize With Positive Vascular Remodeling and Atherosclerotic Plaque Disruption An In Vivo Magnetic Resonance Imaging Study

Background— Local hemodynamic factors, particularly low endothelial shear stress (ESS), play a role in the focal formation of atherosclerosis. We used in vivo MRI to investigate the role of the magnitude of ESS on vascular remodeling, plaque burden, and disruption using a rabbit model of controlled atherothrombosis. Methods and Results— Atherosclerosis was induced in New Zealand white rabbits by cholesterol diet and endothelial denudation. MRI was performed before (pretrigger) and after (posttrigger) inducing plaque disruption with Russell viper venom and histamine. Of the 134 vascular segments studied, 28 contained thrombus (disrupted) and 106 did not (nondisrupted). Disrupted plaques were histologically characterized by a thin, inflamed fibrous cap, a dense lipid core, and mural thrombus. Pretriggered MRI revealed that disrupted plaques clustered at regions with low mean ESS (11.55±5.3 versus 20.9±9.74 dynes/cm2; P<0.001) and low peak ESS (21.5±11.2 versus 49.2±21.5 dynes/cm2; P<0.001) compared with nondisrupted plaques. The peak ESS negatively correlated with the plaque area (r=−0.56, P<0.001) and remodeling ratio (r=−0.4, P=0.008). There was also a negative correlation between the mean ESS and the remodeling ratio (r=−0.55, P<0.001). Both the peak ESS and the mean ESS did not correlate with the % stenosis; there was a weak but statistically significant correlation with the % cross-sectional narrowing (r=0.3, P=0.002 and r=0.2, P=0.04, respectively). Receiver operating characteristic analysis showed that both mean (Area under the curve=0.78; 95% CI, 0.69–0.87) and peak ESS (Area under the curve=0.85; 95% CI, 0.78–0.93) identified disrupted plaques. Conclusions— We demonstrated that low ESS is associated with plaque burden, positive vascular remodeling, and plaque disruption in a rabbit model. Assessment of ESS by noninvasive MRI might be useful for assessing atherosclerotic risk.