Background— In this study, we developed and validated a new approach for in vivo visualization of inflammatory processes by magnetic resonance imaging using biochemically inert nanoemulsions of perfluorocarbons (PFCs). Methods and Results— Local inflammation was provoked in 2 separate murine models of acute cardiac and cerebral ischemia, followed by intravenous injection of PFCs. Simultaneous acquisition of morphologically matching proton ( 1 H) and fluorine ( 19 F) images enabled an exact anatomic localization of PFCs after application. Repetitive 1 H/ 19 F magnetic resonance imaging at 9.4 T revealed a time-dependent infiltration of injected PFCs into the border zone of infarcted areas in both injury models, and histology demonstrated a colocalization of PFCs with cells of the monocyte/macrophage system. We regularly found the accumulation of PFCs in lymph nodes. Using rhodamine-labeled PFCs, we identified circulating monocytes/macrophages as the main cell fraction taking up injected nanoparticles. Conclusions— PFCs can serve as a “positive” contrast agent for the detection of inflammation by magnetic resonance imaging, permitting a spatial resolution close to the anatomic 1 H image and an excellent degree of specificity resulting from the lack of any 19 F background. Because PFCs are nontoxic, this approach may have a broad application in the imaging and diagnosis of numerous inflammatory disease states.
Abstract-The transcription factor signal transducer and activator of transcription 3 (STAT3) participates in a wide variety of physiological processes and directs seemingly contradictory responses such as proliferation and apoptosis. To elucidate its role in the heart, we generated mice harboring a cardiomyocyte-restricted knockout of STAT3 using Cre/loxP-mediated recombination. STAT3-deficient mice developed reduced myocardial capillary density and increased interstitial fibrosis within the first 4 postnatal months, followed by dilated cardiomyopathy with impaired cardiac function and premature death. Conditioned medium from STAT3-deficient cardiomyocytes inhibited endothelial cell proliferation and increased fibroblast proliferation, suggesting the presence of paracrine factors attenuating angiogenesis and promoting fibrosis in vitro. STAT3-deficient mice showed enhanced susceptibility to myocardial ischemia/reperfusion injury and infarction with increased cardiac apoptosis, increased infarct sizes, and reduced cardiac function and survival. Our study establishes a novel role for STAT3 in controlling paracrine circuits in the heart essential for postnatal capillary vasculature maintenance, interstitial matrix deposition balance, and protection from ischemic injury and heart failure. Key Words: mouse Ⅲ signal transduction Ⅲ angiogenesis Ⅲ ischemia Ⅲ heart failure A ctivation of signal transducer and activator of transcription 3 (STAT3) in the heart has been observed in acute myocardial infarction (MI), ischemic preconditioning, and pressure overload. [1][2][3] In this regard, activation of the stressresponsive Janus kinase (JAK)-STAT signaling pathway during ischemia/reperfusion (I/R) injury and MI has been proposed to provide protection against ischemic stress via transcriptional activation of cytoprotective genes. 1,4 Cell culture studies have ascribed some of the cytoprotective actions of the JAK-STAT pathway in cardiomyocytes specifically to STAT3 activation. 5 However, although STAT3 activation is clearly associated with an upregulation of a wide array of target genes in cardiomyocytes, it is unclear which of the reported cardiac responses associated with STAT3 activation are indeed required in vivo for controlling cardiac growth, function, tissue architecture, or protection against cardiovascular stress such as ischemic injury. Importantly, although increased circulating levels of interleukin (IL)-6 -related cytokines predict mortality in patients with heart failure and may enhance gp130 activation in the failing human heart, expression and phosphorylation levels of STAT3 are severely depressed in myocardium obtained from patients with dilated cardiomyopathy, 6 raising the possibility that decreased STAT3 activation may contribute to development of cardiac failure in patients.To elucidate the potential role of STAT3 in cardiac muscle and, in particular, for cardiac protection against physiological and pathophysiological stress, we created mice with a cardiomyocyte-restricted STAT3 deletion. Materials and...
Myoglobin may serve a variety of functions in muscular oxygen supply, such as O 2 storage, facilitated O 2 diffusion, and myoglobin-mediated oxidative phosphorylation. We studied the functional consequences of a myoglobin deficiency on cardiac function by producing myoglobinknockout (myo ؊͞؊ ) mice. )]. These data demonstrate that disruption of myoglobin results in the activation of multiple compensatory mechanisms that steepen the pO 2 gradient and reduce the diffusion path length for O 2 between capillary and the mitochondria; this suggests that myoglobin normally is important for the delivery of oxygen.
Abstract-To investigate the role of adenosine formed extracellularly in vascular homeostasis, mice with a targeted deletion of the cd73/ecto-5Ј-nucleotidase were generated. Southern blot, RT-PCR, and Western blot analysis confirmed the constitutive knockout. In vivo analysis of hemodynamic parameters revealed no significant differences in systolic blood pressure, ejection fraction, or cardiac output between strains. However, basal coronary flow measured in the isolated perfused heart was significantly lower (Ϫ14%; PϽ0.05) in the mutant. Immunohistochemistry revealed strong CD73 expression on the endothelium of conduit vessels in wild-type (WT) mice. Time to carotid artery occlusion after ferric chloride (FeCl 3 ) was significantly reduced by 20% in cd73 Ϫ/Ϫ mice (PϽ0.05). Bleeding time after tail tip resection tended to be shorter in cd73mice (Ϫ35%). In vivo platelet cAMP levels were 0.96Ϯ0.46 in WT versus 0.68Ϯ0.27 pmol/10 6 cells in cd73 Ϫ/Ϫ mice (PϽ0.05). Under in vitro conditions, platelet aggregation in response to ADP (0.05 to 10 mol/L) was undistinguishable between the two strains. In the cremaster model of ischemia-reperfusion, the increase in leukocyte attachment to endothelium was significantly higher in cd73 Ϫ/Ϫ compared with WT littermates (WT 98% versus cd73 Ϫ/Ϫ 245%; PϽ0.005). The constitutive adhesion of monocytes in ex vivo-perfused carotid arteries of WT mice was negligible but significantly increased in arteries of cd73 Ϫ/Ϫ mice (PϽ0.05). Thus, our data provide the first evidence that adenosine, extracellularly formed by CD73, can modulate coronary vascular tone, inhibit platelet activation, and play an important role in leukocyte adhesion to the vascular endothelium in vivo. Key Words: transgenic mice Ⅲ adenosine Ⅲ ecto-5Ј-nucleotidase Ⅲ vascular inflammation Ⅲ thrombosis C D73/ecto-5Ј-nucleotidase, a 70-kDa glycosylphosphatidylinositol (GPI)-anchored cell surface molecule, is expressed on the vascular endothelium and catalyzes the extracellular conversion of 5Ј-AMP to adenosine. 1,2 CD73 is the final step of the extracellular nucleotide breakdown cascade that also involves membrane-associated CD39/ATPdiphosphohydrolase. 3 The product of CD73 is adenosine, a purine nucleoside that has been implicated in many physiological and pathophysiological events. 4 There are four known G-coupled adenosine receptors: A 1 , A 2A , A 2B , and A 3 , each of which operates via different intracellular signaling mechanisms and exhibits distinct patterns of tissue distribution. 5 In human neutrophils, adenosine A 1 and A 2 receptor occupancy mediate opposing roles for adenosine in inflammation: A 1 activation is proinflammatory, whereas the A 2 receptor plays an anti-inflammatory role. 6 A 2 receptor activation inhibits the neutrophil oxidative burst, whereas the A 3 receptor inhibits neutrophil degranulation 7 and may play an important role in inflammation by inhibiting eosinophil migration. 8 Recently, deletion of the A 2A receptor in transgenic mice revealed that this receptor is critical for the limitation a...
Abstract-For the specific analysis of endothelial NO synthase (eNOS) function in the coronary vasculature, we generated a mouse homozygous for a defective eNOS gene (eNOSϪ/Ϫ). Western blot as well as immunohistochemical staining revealed the absence of eNOS protein in eNOSϪ/Ϫ mice. Aortic endothelial cells derived from eNOSϪ/Ϫ mice displayed only background levels of NO x formation compared with wild-type (WT) cells (88 versus 1990 pmol). eNOSϪ/Ϫ mice were hypertensive (mean arterial pressure, 135Ϯ15 versus 107Ϯ8 mm Hg in WT) without the development of cardiac hypertrophy. Coronary hemodynamics, analyzed in Langendorff-perfused hearts, showed no differences either in basal coronary flow or in maximal and repayment flow of reactive hyperemia. Acute NOS inhibition with N -nitro-L-arginine methyl ester (L-NAME) in WT hearts substantially reduced basal flow and reactive hyperemia. The coronary response to acetylcholine (ACh) (500 nmol/L) was biphasic: An initial vasoconstriction (flow, Ϫ35%) in WT hearts was followed by sustained vasodilation (ϩ190%). L-NAME significantly reduced vasodilation in WT hearts (ϩ125%) but did not alter the initial vasoconstriction. In eNOSϪ/Ϫ hearts, the initial vasoconstriction was augmented (Ϫ70%), whereas the ACh-induced vasodilation was not affected. Inhibition of cyclooxygenase with diclofenac converted the ACh-induced vasodilation into vasoconstriction (Ϫ49% decrease of basal flow). This effect was even more pronounced in eNOSϪ/Ϫ hearts (Ϫ71%). Our results demonstrate that (1) acute inhibition of eNOS reveals a role for NO in setting the basal coronary vascular tone as well as participation in reactive hyperemia and the response to ACh; (2) chronic inhibition of NO formation in eNOSϪ/Ϫ mutant mice induces no changes in basal coronary flow and reactive hyperemia, suggesting the activation of important compensatory mechanisms; and (3) Key Words: heart Ⅲ gene targeting Ⅲ reactive hyperemia Ⅲ coronary flow Ⅲ blood pressure E ndothelial NO synthase, also called type III NO synthase, is the major NOS isoenzyme that is widely expressed in endothelial cells throughout the vascular bed. It is generally accepted that endothelium-derived NO is an important factor in the control of basal vascular tone.1 NO is also involved in receptor-mediated vasodilation in response to various agonists such as ACh, ATP, thrombin, bradykinin, and others. Through experiments using NOS inhibitors 2,3 but also by use of genetically modified animals, 4,5 it has been demonstrated that functional inactivation of eNOS activity results in hypertension. In addition to the control of vascular tone, NO inhibits platelet aggregation and leukocyte adhesion to the vessel wall as well as proliferation and migration of smooth muscle cells. Thus, eNOS is considered to play an important role in maintaining the antiatherogenic surface of the vessel wall. 6In the heart, eNOS is expressed primarily in the coronary and endocardial endothelia. In addition, eNOS has been localized to cardiac myocytes and the specialized cells of s...
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