Search

Objective: Natriuretic peptide receptor-C (NPR-C/NPR-3) is a cell surface protein involved in vascular remodelling that is up-regulated in atherosclerosis. NPR-C expression has not been well characterized in human carotid artery occlusive lesions. We hypothesized that NPR-C expression correlates with intimal features of vulnerable atherosclerotic carotid artery plaque. Methods: To test this hypothesis, we evaluated NPR-C expression by immunohistochemistry (IHC) in carotid endarterectomy (CEA) specimens isolated from 18 patients. The grade, location, and co-localization of NPR-C in CEA specimens were evaluated using two tissue analysis techniques. Results: Relative to minimally diseased CEA specimens, we observed avid NPR-C tissue staining in the intima of maximally diseased CEA specimens (65%; p=0.06). Specifically, maximally diseased CEA specimens demonstrated increased NPR-C expression in the superficial intima (61%, p=0.17), and deep intima (138% increase; p=0.05). In the superficial intima, NPR-C expression significantly co-localized with vascular smooth muscle cells (VSMCs) and macrophages. The intensity of NPR-C expression was also higher in the superficial intima plaque shoulder and cap regions, and significantly correlated with atheroma and fibroatheroma vulnerable plaque regions (β=1.04, 95% CI=0.46, 1.64). Conclusion: These findings demonstrate significant NPR-C expression in the intima of advanced carotid artery plaques. Furthermore, NPR-C expression was higher in vulnerable carotid plaque intimal regions, and correlate with features of advanced disease. Our findings suggest that NPR-C may serve as a potential biomarker for carotid plaque vulnerability and progression, in patients with advanced carotid artery occlusive disease.

Ischemia-related processes associated with the generation of inflammatory molecules such as reactive oxygen species (ROS) are difficult to detect at the acute stage before the physiologic and anatomic evidence of tissue damage is present. Evaluation of the inflammatory and healing response early after an ischemic event in vivo will aid in treatment selection and patient outcomes. We introduce a novel near-infrared hydrocyanine molecular probe for the detection of ROS as a marker of tissue response to ischemia and a precursor to angiogenesis and remodeling. The synthesized molecular probe, initially a non-fluorescent hydrocyanine conjugated to polyethylene glycol, converts to a highly fluorescent cyanine reporter upon oxidation.The probe was applied in a preclinical mouse model for myocardial infarction, where ligation and removal of a portion of the femoral artery in the hindlimb resulted in temporary ischemia followed by angiogenesis and healing.The observed increase in fluorescence intensity was approximately sixfold over 24 h in the ischemic tissue relative to the uninjured control limb and was attributed to the higher concentration of ROS in the ischemic tissue.These results demonstrate the potential for non-invasive sensing for interrogating the inflammatory and healing response in ischemic tissue.

A new 17O-labeled blood contrast agent was injected intravenously in control dogs. Electrocardiogram (ECG)-triggered myocardial T1ρ imaging was performed to obtain spin-locking T1ρ-weighted myocardial signals for the detection of resultant metabolite H217O water in the heart. Bolus and slow injection methods of various doses of the 17O-labeled and 16O-labeled agents were carried out in order to evaluate the sensitivity of this method and determine the optimal injection method. Bolus injection provided approximately 1% signal reduction, whereas slow injection with larger amount of agent yielded 11.9 ± 0.6% signal reduction. Myocardial oxygen consumption rate was determined by a technique to quantify cerebral oxygenation consumption rate previously developed in 17O brain studies. With either injection method, myocardial oxygen consumption rate at rest was 5.0 – 5.6 μmol/g/min. Therefore, it appears feasible to detect metabolically generated HO water in vivo in the heart, using the 17O-labeled blood tracer. Myocardial oxygen consumption rate can then be quantified in vivo, which may open new doors for the assessment of myocardial metabolism. Magn Reson Med 63:1442–1447, 2010. © 2010 Wiley-Liss, Inc.

Activation of phospholipases leads to the release of arachidonic acid and lysophospholipids that play prominent roles in regulating vasomotor tone. To identify the role of calcium-independent phospholipase A2β (iPLA2β) in vasomotor function, we measured vascular responses to phenylephrine (PE) and ACh in mesenteric arterioles from wild-type (WT; iPLA2β+/+) mice and those lacking the β-isoform (iPLA2β−/−) both ex vivo and in vivo. Vessels isolated from iPLA2β−/−mice demonstrated increased constriction to PE, despite lower basal smooth muscle calcium levels, and decreased vasodilation to ACh compared with iPLA2β+/+mice. PE constriction resulted in initial intracellular calcium release with subsequent steady-state constriction that depended on extracellular calcium influx. Endothelial denudation had no effect on vessel tone or PE-induced constriction although the dilation to ACh was significantly reduced in iPLA2β+/+vessels. In contrast, vessels from iPLA2β−/−constricted by 54% after denudation, indicating smooth muscle hypercontractility. In vivo, blood pressure, resting vessel diameter, and constriction of mesenteric vessels to PE were not different in iPLA2β−/−vessels compared with WT mouse vessels. However, relaxation after ACh administration in situ was attenuated, indicating an endothelial inability to induce dilation in response to ACh. In cultured endothelial cells, inhibition of iPLA2β with ( S)-(E)-6-(bromomethylene)tetrahydro-3-(1-naphthalenyl)-2 H-pyran-2-one (BEL) decreased endothelial nitric oxide synthase phosphorylation and reduced endothelial agonist-induced intracellular calcium release as well as extracellular calcium influx. We conclude that iPLA2β is an important mediator of vascular relaxation and intracellular calcium homeostasis in both smooth muscle and endothelial cells and that ablation of iPLA2β causes agonist-induced smooth muscle hypercontractility and reduced agonist-induced endothelial dilation.

Background— A comprehensive evaluation of myocardial ischemia requires measures of both oxygen supply and demand. Positron emission tomography (PET) is currently the gold standard for such evaluations, but its use is limited because of its ionizing radiation, limited availability, and high cost. A cardiac MRI method was developed for assessing myocardial oxygenation. The purpose of this study was to evaluate and validate this technique compared with PET during pharmacological stress in a canine model of coronary artery stenosis. Methods and Results— Twenty-one beagles and small mongrel dogs without coronary artery stenosis (controls) or with moderate to severe acute coronary artery stenosis underwent MRI and PET imaging at rest and during dipyridamole vasodilation or dobutamine stress to induce a wide range of changes in cardiac perfusion and oxygenation. MRI first-pass perfusion imaging was performed to quantify myocardial blood flow and volume. The MRI blood oxygen level-dependent technique was used to determine the myocardial oxygen extraction fraction during pharmacological hyperemia. Myocardial oxygen consumption was determined by the Fick law. In the same dogs, 15 O-water and 11 C-acetate were used to measure myocardial blood flow and myocardial oxygen consumption, respectively, by PET. Regional assessments were performed for both MR and PET. MRI data correlated nicely with PET values for myocardial blood flow ( R 2 =0.79, P R 2 =0.74, P R 2 =0.66, P Conclusions— Cardiac MRI methods may provide an alternative to radionuclide imaging in settings of myocardial ischemia. Our newly developed quantitative MRI oxygenation imaging technique may be a valuable noninvasive tool to directly evaluate myocardial energetics and efficiency.

A biodegradable positron-emitting dendritic nanoprobe targeted at α v β 3 integrin, a biological marker known to modulate angiogenesis, was developed for the noninvasive imaging of angiogenesis. The nanoprobe has a modular multivalent core-shell architecture consisting of a biodegradable heterobifunctional dendritic core chemoselectively functionalized with heterobifunctional polyethylene oxide (PEO) chains that form a protective shell, which imparts biological stealth and dictates the pharmacokinetics. Each of the 8 branches of the dendritic core was functionalized for labeling with radiohalogens. Placement of radioactive moieties at the core was designed to prevent in vivo dehalogenation, a potential problem for radiohalogens in imaging and therapy. Targeting peptides of cyclic arginine–glycine–aspartic acid (RGD) motifs were installed at the terminal ends of the PEO chains to enhance their accessibility to α v β 3 integrin receptors. This nanoscale design enabled a 50-fold enhancement of the binding affinity to α v β 3 integrin receptors with respect to the monovalent RGD peptide alone, from 10.40 nM to 0.18 nM IC 50 . Cell-based assays of the 125 I-labeled dendritic nanoprobes using α v β 3 -positive cells showed a 6-fold increase in α v β 3 receptor-mediated endocytosis of the targeted nanoprobe compared with the nontargeted nanoprobe, whereas α v β 3 -negative cells showed no enhancement of cell uptake over time. In vivo biodistribution studies of 76 Br-labeled dendritic nanoprobes showed excellent bioavailability for the targeted and nontargeted nanoprobes. In vivo studies in a murine hindlimb ischemia model for angiogenesis revealed high specific accumulation of 76 Br-labeled dendritic nanoprobes targeted at α v β 3 integrins in angiogenic muscles, allowing highly selective imaging of this critically important process.

Recently, we have identified the dramatic depletion of cardiolipin (CL) in diabetic myocardium 6 weeks after streptozotocin (STZ) injection that was accompanied by increases in triacylglycerol content and multiple changes in polar lipid molecular species. However, after 6 weeks in the diabetic state, the predominant lipid hallmarks of diabetic cardiomyopathy were each present concomitantly, and thus, it was impossible to identify the temporal course of lipid alterations in diabetic myocardium. Using the newly developed enhanced shotgun lipidomics approach, we demonstrated the dramatic loss of abundant CL molecular species in STZ-treated hearts at the very earliest stages of diabetes accompanied by a profound remodeling of the remaining CL molecular species including a 16-fold increase in the content of 18:2-22:6-22:6-22:6 CL. These alterations in CL metabolism occur within days after the induction of the diabetic state and precede the triacylglycerol accumulation manifest in diabetic myocardium. Similarly, in ob/ob mice, a dramatic and progressive redistribution from 18:2 FA-containing CL molecular species to 22:6 FA-containing CL molecular species was also identified. Collectively, these results demonstrate alterations in CL hydrolysis and remodeling at the earliest stages of diabetes and are consistent with a role for alterations in CL content in precipitating mitochondrial dysfunction in diabetic cardiomyopathy.

Objectives. This study was designed to define the extent of inhibition of thrombin activity achieved with conjunctive fixed dose intravenous sodium heparin compared with fixed dose subcutaneous calcium heparin in patients receiving intravenous streptokinase for acute myocardial infarction. Background. The role of heparin therapy during coronary thrombolysis with streptokinase is controversial, in part because the efficacy of different conjunctive heparin regimens in inhibiting early increases of thrombin activity is not known. Methods. Twenty-eight patients treated with 1.5 million U of streptokinase and 165 mg of aspirin for acute myocardial infarction were randomly assigned to receive fixed dose subcutaneous heparin therapy (12,500 U every 12 h delayed until 4 h after the end of streptokinase therapy [n = 14]) or fixed dose intravenous heparin (5,000-U bolus followed by 1,000-U/h infusion [n = 14]). Anticoagulation was assessed with serial measurements of activated partial thromboplastin time, and thrombin activity by measuring fibrinopeptide A and thrombin-antithrombin III complex levels. Plasma concentrations of creatine kinase (CK) MM isoforms were measured for 3 h to determine recanalization (increase la activity > 0.18%/min). Results. Recanalization occurred in 27%, 64% and 79% of patients given subcutaneous heparin versus 43%, 76% and 86% of those given intravenous heparin at 1, 2 and 3 h, respectively (p = 0.6). Concentrations of fibrinopeptide A (mean ± SEM) at 1 h were higher in patients without (n = 5) than in those with (n = 23) CK-MM isofona criteria for recanalization (76.4 ± 25.7 vs. 25.2 ± 52 nmol/liter, p = 0.02), and at 1, 2 and 3 h were significantly lower with fixed dose intravenous heparin (18.4 ± 4.8 vs. 46.7 ± 102 nmol/liter at 1 h, p = 0.004) than without heparin. After fixed dose subcutaneous heparin at 4 h, fibrinopeptide A levels were similar in both groups despite lower activated partial thromboplastin times in patients who received fixed dose subcutaneous heparin. However, fibrinopeptide A was not consistently suppressed in either group (fixed dose subcutaneous heparin 8.7 ± 1.8 nmol/liter vs. fixed dose intravenous heparin 11.8 ± 5.2 nmol/liter) at 48 h (p = 0.4). No significant changes in the concentration of thrombin-antithrombin III complexes were found between the two groups. Conclusions. Fixed dose intravenous heparin attenuates increases in fibrinopeptide A early after streptokinase. Subsequent fixed dose intravenous and subcutaneous heparin have similar effects but are relatively ineffective in suppressing thrombin activity, suggesting a role for more potent antithrombin agents during coronary thrombolysis with streptokinase.

ObjectivesWe sought to determine whether brief, profound inhibition of thrombin or prothrombin activation by factor Xa limits neointimal formation and stenosis after arterial injury.BackgroundThrombin has been implicated as a mediator of neointimal formation, but adjunctive administration of anticoagulant agents has not proven effective to decrease restenosis in patients undergoing coronary angioplasty.MethodsWe infused recombinant desulfatohirudin (r-hirudin, bolus of 2 mg/kg body weight followed by 2 mg/kg per h, n = 9), heparin (100 U/kg per h, n = 6) or recombinant tick anticoagulant peptide (rTAP, 1-mg/kg bolus followed by 3 mg/kg per h, n = 5), a specific inhibitor of factor Xa, intravenously, beginning 15 min before and for up to 3 h after repetitive balloon hyperinflations sufficient to disrupt the internal elastic lamina in a carotid artery of minipigs with hypercholesterolemia induced by feeding them an atherogenic diet.ResultsPartial thromboplastin time was increased six- to sevenfold over baseline levels at the end of the infusions of the anticoagulant agents. Lumen stenosis measured histologically 4 weeks after balloon-induced carotid injury was 29 ± 16% (mean ± SEM) in r-hirudin-treated, 52 ± 19% in rTAP-treated and 76 ± 18% in heparin-treated pigs (p < 0.02 for r-hirudin vs. heparin treatment).ConclusionsThe marked reduction of stenosis in r-hirudin-treated animals indicates that thrombin plays a major role in neointimal formation after balloon-induced arterial injury. A relatively brief interval of profound, direct inhibition of thrombin may be particularly effective to attenuate restenosis after balloon angioplasty.(J Am Coll Cardiol 1996;28:1849–55) ©1996 by the American College of Cardiology

Coronary thrombolysis in patients frequently unmasks high grade residual stenosis. To determine whether beneficial effects of reperfusion are compromised by critical residual coronary stenosis, 14 dogs were instrumented with an external left anterior descending coronary artery balloon occluder, Doppler flow probe and adjustable screw clamp. In eight of the dogs, critical stenosis (abolition of reactive hyperemia after a 20 s occlusion; 95.7 +/- 1.0% cross-sectional area reduction) was induced before occlusion and maintained. In the control group (n = 6), no stenosis was induced. Each dog was subjected to 2 h of myocardial ischemia followed by balloon deflation and 24 h of reperfusion. Myocardial blood flow assessed with microspheres was similar during balloon inflation in both groups and indicative of profound ischemia. Transmural blood flow to the reperfused zone assessed 1 min after balloon deflation was significantly greater in control dogs without residual stenosis (383% of normal compared with 120% of normal in dogs with stenosis) (p less than 0.01). Compromise of transmural flow persisted in dogs with stenosis (85% compared with 121% of normal in control dogs after 1 h, p less than 0.05; and 49% compared with 68% after 24 h of reperfusion, p less than 0.05). Diminution of subendocardial blood flow after reperfusion was particularly marked. The extent of infarction was greater in the heart of dogs with residual stenosis. Thus, residual critical coronary stenosis compromises nutritional perfusion and salvage of reperfused myocardium after recanalization. These observations underscore the need for prompt identification of patients with high grade residual stenosis early after coronary thrombolysis and the potential value of angioplasty or coronary surgery in selected patients soon after initial recanalization.

Purpose: We evaluated the contributions of coagulation factors IIa (thrombin) and Xa to small-diameter prosthetic graft thrombogenicity in vivo. Methods: Preclotted and nonpreclotted (collagen-coated) polyester grafts were studied before and 24 hours after implantation into pig femoral arteries. After incubation of explanted grafts was performed with plasma depleted of vitamin K-dependent coagulation factors by barium chloride adsorbtion (Ba-plasma), graft-associated thrombin activity was determined by radioimmunoassay for fibrinopeptide A. Fibrinopeptide A levels reflect thrombin-mediated fibrin formation. Factor Xa activity was characterized by measuring activation of prothrombin added to Ba-plasma. Results: Thrombin and factor Xa were associated with the luminal surfaces of preclotted grafts before and 24 hours after implantation. Nonpreclotted grafts had negligible procoagulant activity before implantation. After 24 hours in vivo graft-associated factor Xa activity was similar in both nonpreclotted and preclotted grafts; however, more thrombin was bound to nonpreclotted coated grafts ( p Conclusions: The procoagulant activity of small-diameter prosthetic grafts persists for 24 hours after implantation and is attributable not only to graft-associated thrombin but also to de novo thrombin elaboration induced by factor Xa. Moreover, graft-associated procoagulant activity is not dependent on preclotting because it develops on nonpreclotted, collagen-coated grafts as well. Treatment strategies to attenuate graft thrombosis may require the inhibition of both thrombin and factor Xa.

Background Impaired myocardial oxygenation leading to ischemia is central to the pathophysiology of coronary artery disease and an important contributor to other common cardiovascular disease conditions such as left ventricular hypertrophy, dilated cardiomyopathy, renal heart disease and valvular heart disease. Positron Emission Tomography (PET) can quantify myocardial oxygen consumption (MVO2), but has limited applications due to its relatively low spatial resolution, high cost, and ionizing radiation. Purpose

Purpose Fluid shear stress was thought to lead to atherosclerotic plaque progression, but such measurement procedures are often invasive. The goal of the present study is to address the biomechanical pathways of atherosclerosis progression and regression with our non-invasive MRI methods and a 3D fluid-structure interaction (FSI) model. The study was performed in a large animal atherosclerotic model.