Christoph Jacoby

In Vivo Monitoring of Inflammation After Cardiac and Cerebral Ischemia by Fluorine Magnetic Resonance Imaging

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 (1H) and fluorine (19F) images enabled an exact anatomic localization of PFCs after application. Repetitive 1H/19F 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 1H image and an excellent degree of specificity resulting from the lack of any 19F background. Because PFCs are nontoxic, this approach may have a broad application in the imaging and diagnosis of numerous inflammatory disease states.

Taurine transporter knockout depletes muscle taurine levels and results in severe skeletal muscle impairment but leaves cardiac function uncompromised

Taurine is the most abundant free amino acid in heart and skeletal muscle. In the present study, the effects of hereditary taurine deficiency on muscle function were examined in taurine transporter knockout (taut−/−) mice. These mice show an almost complete depletion of heart and skeletal muscle taurine levels. Treadmill experiments demonstrated that total exercise capacity of taut−/− mice was reduced by >80% compared with wild‐type controls. The decreased performance of taut−/− mice correlated with increased lactate levels in serum during exercise. Surprisingly, cardiac function of taut−/− mice as assessed by magnetic resonance imaging, echocardiography, and isolated heart studies showed a largely normal phenotype under both control and stimulated conditions. However, analysis of taut−/− skeletal muscle revealed electromyographic abnormalities. 1H nuclear magnetic resonance spectroscopy of tissue extracts showed that in the heart of taut−/− mice the lack of taurine was compensated by the up‐regulation of various organic solutes. In contrast, a deficit of >10 mM in total organic osmolyte concentration was found in skeletal muscle. The present study identifies taurine transport as a crucial factor for the maintenance of skeletal muscle function and total exercise capacity, while cardiac muscle apparently can compensate for the loss of taurine.

RIP3, a kinase promoting necroptotic cell death, mediates adverse remodelling after myocardial infarction.

AIMS Programmed necrosis (necroptosis) represents a newly identified mechanism of cell death combining features of both apoptosis and necrosis. Like apoptosis, necroptosis is tightly regulated by distinct signalling pathways. A key regulatory role in programmed necrosis has been attributed to interactions of the receptor-interacting protein kinases, RIP1 and RIP3. However, the specific functional role of RIP3-dependent signalling and necroptosis in the heart is unknown. The aims of this study were thus to assess the significance of necroptosis and RIP3 in the context of myocardial ischaemia. METHODS AND RESULTS Immunoblots revealed strong expression of RIP3 in murine hearts, indicating potential functional significance of this protein in the myocardium. Consistent with a role in promoting necroptosis, adenoviral overexpression of RIP3 in neonatal rat cardiomyocytes and stimulation with TNF-α induced the formation of a complex of RIP1 and RIP3. Moreover, RIP3 overexpression was sufficient to induce necroptosis of cardiomyocytes. In vivo, cardiac expression of RIP3 was up-regulated upon myocardial infarction (MI). Conversely, mice deficient for RIP3 (RIP3(-/-)) showed a significantly better ejection fraction (45 ± 3.6 vs. 32 ± 4.4%, P < 0.05) and less hypertrophy in magnetic resonance imaging studies 30 days after experimental infarction due to left anterior descending coronary artery ligation. This was accompanied by a diminished inflammatory response of infarcted hearts and decreased generation of reactive oxygen species. CONCLUSION Here, we show that RIP3-dependent necroptosis modulates post-ischaemic adverse remodelling in a mouse model of MI. This novel signalling pathway may thus be an attractive target for future therapies that aim to limit the adverse consequences of myocardial ischaemia.

Biglycan Is Required for Adaptive Remodeling After Myocardial Infarction

Background— After myocardial infarction (MI), extensive remodeling of extracellular matrix contributes to scar formation and preservation of hemodynamic function. On the other hand, adverse and excessive extracellular matrix remodeling leads to fibrosis and impaired function. The present study investigates the role of the small leucine-rich proteoglycan biglycan during cardiac extracellular matrix remodeling and cardiac hemodynamics after MI. Methods and Results— Experimental MI was induced in wild-type (WT) and bgn−/0 mice by permanent ligation of the left anterior descending coronary artery. Biglycan expression was strongly increased at 3, 7, and 14 days after MI in WT mice. bgn−/0 mice showed increased mortality rates after MI as a result of frequent left ventricular (LV) ruptures. Furthermore, tensile strength of the LV derived from bgn−/0 mice 21 days after MI was reduced as measured ex vivo. Collagen matrix organization was severely impaired in bgn−/0 mice, as shown by birefringence analysis of Sirius red staining and electron microscopy of collagen fibrils. At 21 days after MI, LV hemodynamic parameters were assessed by pressure-volume measurements in vivo to obtain LV end-diastolic pressure, end-diastolic volume, and end-systolic volume. bgn−/0 mice were characterized by aggravated LV dilation evidenced by increased LV end-diastolic volume (bgn−/0, 111±4.2 &mgr;L versus WT, 96±4.4 &mgr;L; P<0.05) and LV end-diastolic pressure (bgn−/0, 24±2.7 versus WT, 18±1.8 mm Hg; P<0.05) and severely impaired LV function (EF, bgn−/0, 12±2% versus WT, 21±4%; P<0.05) 21 days after MI. Conclusion— Biglycan is required for stable collagen matrix formation of infarct scars and for preservation of cardiac hemodynamic function.

Early Assessment of Pulmonary Inflammation by 19F MRI In Vivo

Background—Emulsified perfluorocarbons (PFCs) are preferentially phagocytized by monocytes/macrophages and are readily detected by 19F MRI. This study tests the hypothesis that 19F MRI can be used to quantitate pulmonary inflammation by tracking of infiltrating PFC-loaded monocytes. Methods and Results—Pneumonia was induced in mice by intratracheal instillation of lipopolysaccharides (LPS) followed by intravenous injection of PFCs. Whereas regular 1H MRI provided no evidence of lung injury 24 hours after LPS, the concurrent 19F images clearly show PFC accumulation in both pulmonary lobes. Imaging at 48 hours after LPS revealed signals in 1H images at the same location as the 24-hour 19F signals. Thus, progressive pneumonia was first predicted by 19F MRI early after PFC administration. Without LPS, at no time were 19F signals observed within the lung. Histology and fluorescence-activated cell sorting (FACS) combined with 19F MRI confirmed the presence of infiltrating PFC-loaded monocytes/macrophages after LPS challenge. Additional experiments with graded doses of LPS demonstrated that 19F signal intensity strongly correlated with both LPS dose and pathological markers of lung inflammation. In separate studies, dexamethasone and CGS21680 (adenosine 2A receptor agonist) were used to demonstrate the ability of 19F MRI to monitor anti-inflammatory therapies. Conclusions—PFCs serve as a contrast agent for the prognostic and quantitative assessment of pulmonary inflammation by in vivo 19F MRI, which is characterized by a high degree of specificity due to the lack of any 19F background. Because PFCs are biochemically inert, this approach may also be suitable for human applications.

The natriuretic peptide/guanylyl cyclase--a system functions as a stress-responsive regulator of angiogenesis in mice.

Cardiac atrial natriuretic peptide (ANP) and B-type natriuretic peptide (BNP) modulate blood pressure and volume by activation of the receptor guanylyl cyclase-A (GC-A) and subsequent intracellular cGMP formation. Here we report what we believe to be a novel function of these peptides as paracrine regulators of vascular regeneration. In mice with systemic deletion of the GC-A gene, vascular regeneration in response to critical hind limb ischemia was severely impaired. Similar attenuation of ischemic angiogenesis was observed in mice with conditional, endothelial cell-restricted GC-A deletion (here termed EC GC-A KO mice). In contrast, smooth muscle cell-restricted GC-A ablation did not affect ischemic neovascularization. Immunohistochemistry and RT-PCR revealed BNP expression in activated satellite cells within the ischemic muscle, suggesting that local BNP elicits protective endothelial effects. Since within the heart, BNP is mainly induced in cardiomyocytes by mechanical load, we investigated whether the natriuretic peptide/GC-A system also regulates angiogenesis accompanying load-induced cardiac hypertrophy. EC GC-A KO hearts showed diminished angiogenesis, mild fibrosis, and diastolic dysfunction. In vitro BNP/GC-A stimulated proliferation and migration of cultured microvascular endothelia by activating cGMP-dependent protein kinase I and phosphorylating vasodilator-stimulated phosphoprotein and p38 MAPK. We therefore conclude that BNP, produced by activated satellite cells within ischemic skeletal muscle or by cardiomyocytes in response to pressure load, regulates the regeneration of neighboring endothelia via GC-A. This paracrine communication might be critically involved in coordinating muscle regeneration/hypertrophy and angiogenesis.

Selective Activation of Adenosine A2A Receptors on Immune Cells by a CD73-Dependent Prodrug Suppresses Joint Inflammation in Experimental Rheumatoid Arthritis

Phosphorylated adenosine A2A receptor agonists suppressed inflammation in a model of arthritis without A2A-mediated vasodilatory side effects. Separating the Wheat from the Chaff Extolling the virtues of simple building design, the modern architect Ludwig Mies van der Rohe famously declared that “less is more,” a philosophy that applies to modern drug design as well. Because simpler drugs have fewer side effects, the promise of adenosine A2A receptor agonists as therapeutics would grow if one could only separate their anti-inflammatory and vasodilator functions. Now, Flögel et al. built an adenosine A2A receptor agonist (chet-AMP) that displays only the anti-inflammatory function in an animal model of rheumatoid arthritis. How the authors accomplished this feat lies “in the details,” to again paraphrase van der Rohe. To isolate the anti-inflammatory effects of A2AR, Flögel et al. synthesized a prodrug that required, for its activation, the presence of ecto-5′-nucleotidase (CD73), which is mainly found on endothelial and immune cells. Using 19F magnetic resonance imaging to track inflammation noninvasively over time, the authors showed that chet-AMP, but not chet-adenosine, reduced inflammation in a mouse model of collagen-induced arthritis. This effect was dependent on the presence of both CD73 and A2AR, and no vasodilation was observed until drug concentrations were increased 100-fold. Physicians most often use corticosteroids to treat inflammatory conditions, but these drugs, although effective, can cause serious complications. By simplifying delivery of a drug only to the sites where it is needed most, Flögel et al. quell inflammation and avoid an undesirable side effect. Adenosine A2A receptor (A2AR) agonists are both highly effective anti-inflammatory agents and potent vasodilators. To separate these two activities, we have synthesized phosphorylated A2AR agonists (prodrugs) that require the presence of ecto-5′-nucleotidase (CD73) to become activated. In the model of collagen-induced arthritis, 2-(cyclohexylethylthio)adenosine 5′-monophosphate (chet-AMP), but not 2-(cyclohexylethylthio)adenosine (chet-adenosine), potently reduced inflammation as assessed by fluorine-19 (19F) magnetic resonance imaging and by histology. The prodrug effect was blunted by inhibition of CD73 and A2AR. The selectivity of drug action is due to profound up-regulation of CD73 and adenosine A2AR expression in neutrophils and inflammatory monocytes as found in recovered cells from the synovial fluid of arthritic mice. Plasma chet-adenosine was in the subnanomolar range when chet-AMP was applied, whereas concentrations required for vasodilation were about 100 times higher. Thus, chet-AMP is a potent immunosuppressant with negligible vasodilatory activity. These data suggest that phosphorylated A2AR agonists may serve as a promising new group of drugs for targeted immunotherapy of inflammation.

Probing different perfluorocarbons for in vivo inflammation imaging by 19F MRI: image reconstruction, biological half-lives and sensitivity.

Inflammatory processes can reliably be assessed by 19F MRI using perfluorocarbons (PFCs), which is primarily based on the efficient uptake of emulsified PFCs by circulating cells of the monocyte–macrophage system and subsequent infiltration of the 19F‐labeled cells into affected tissue. An ideal candidate for the sensitive detection of fluorine‐loaded cells is the biochemically inert perfluoro‐15‐crown‐5 ether (PFCE), as it contains 20 magnetically equivalent 19F atoms. However, the biological half‐life of PFCE in the liver and spleen is extremely long, and so this substance is not suitable for future clinical applications. In the present study, we investigated alternative, nontoxic PFCs with predicted short biological half‐lives and high fluorine content: perfluorooctyl bromide (PFOB), perfluorodecalin (PFD) and trans‐bis‐perfluorobutyl ethylene (F‐44E). Despite the complex spectra of these compounds, we obtained artifact‐free images using sine‐squared acquisition‐weighted three‐dimensional chemical shift imaging and dedicated reconstruction accomplished with in‐house‐developed software. The signal‐to‐noise ratio of the images was maximized using a Nutall window with only moderate localization error. Using this approach, the retention times of the different PFCs in murine liver and spleen were determined at 9.4 T. The biological half‐lives were estimated to be 9 days (PFD), 12 days (PFOB) and 28 days (F‐44E), compared with more than 250 days for PFCE. In vivo sensitivity for inflammation imaging was assessed using an ear clip injury model. The alternative PFCs PFOB and F‐44E provided 37% and 43%, respectively, of the PFCE intensities, whereas PFD did not show any signal in the ear model. Thus, for in vivo monitoring of inflammatory processes, PFOB emerges as the most promising candidate for possible future translation of 19F MR inflammation imaging to human applications. Copyright

Direct comparison of magnetic resonance imaging and conductance microcatheter in the evaluation of left ventricular function in mice

Abstract The aim of the present work was to study the reliability of conductance microcatheter volumetric measurements as compared to magnetic resonance imaging (MRI) in the same set of mice. Mice left ventricular (LV) volumes were monitored under basal conditions and in a hypertrophy model induced by transverse aortic constriction (TAC). Cardiac function was evaluated in isoflurane anesthetized mice (n = 8) by MRI followed by 1.4 F Millar microtip catheter measurements. The second group of mice with TACinduced cardiac hypertrophy was studied eight weeks after surgery. Reliability of 3D–reconstructed MRI data was confirmed by comparison with autopsy masses (autopsy LV mass = 73.6 ± 3.4 mg; MRI LV mass = 76.9 ± 3.7 mg). Conduction catheter was found to greatly underestimate end–diastolic and end–systolic volumes and thus stroke volume as well as cardiac output in control mice (MRI: EDV = 79 ± 8 µl, ESV = 27±9 µl, SV = 51 ± 9 µl, CO = 25 ± 6 ml/min; Catheter: EDV = 28 ± 5 µl, ESV = 8 ± 4 µl, SV = 19 ± 4 µl, CO = 10 ± 2 ml/min). However, values for ejection fraction showed no significant differences between the two methods. In the hypertrophy model, stroke volume and cardiac output were increased when measured with MRI (SV: +19 ± 20%; CO: +28 ± 27%), whereas catheter data showed opposite directional changes (SV: –22 ± 37%; CO: –31 ± 37%). Ejection fraction was found to be reduced only in catheter measurements (–31 ± 26%). In summary, our data demonstrate that absolute volumetric values are strikingly underestimated by conduction catheter measurements and that even detection of directional changes with this method may not always be feasible.

Lack of Myoglobin Causes a Switch in Cardiac Substrate Selection

Myoglobin is an important intracellular O2 binding hemoprotein in heart and skeletal muscle. Surprisingly, disruption of myoglobin in mice (myo−/−) resulted in no obvious phenotype and normal cardiac function was suggested to be mediated by structural alterations that tend to steepen the oxygen pressure gradient from capillary to mitochondria. Here we report that lack of myoglobin causes a biochemical shift in cardiac substrate utilization from fatty acid to glucose oxidation. Proteome and gene expression analysis uncovered key enzymes of mitochondrial β-oxidation as well as the nuclear receptor PPARα to be downregulated in myoglobin-deficient hearts. Using FDG-PET we showed a substantially increased in vivo cardiac uptake of glucose in myo−/− mice (6.7±2.3 versus 0.8±0.5% of injected dose in wild-type, n=5, P<0.001), which was associated with an upregulation of the glucose transporter GLUT4. The metabolic switch was confirmed by 13C NMR spetroscopic isotopomer studies of isolated hearts which revealed that [1,6-13C2]glucose utilization was increased in myo−/− hearts (38±8% versus 22±5% in wild-type, n=6, P<0.05), and concomitantly, [U-13C16]palmitate utilization was decreased in the myoglobin-deficient group (42±6% versus 63±11% in wild-type, n=6, P<0.05). Because of the O2-sparing effect of glucose utilization, the observed shift in substrate metabolism benefits energy homoeostasis and therefore represents a molecular adaptation process allowing to compensate for lack of the cytosolic oxygen carrier myoglobin. Furthermore, our data suggest that an altered myoglobin level itself may be a critical determinant for substrate selection in the heart. The full text of this article is available online at http://circres.ahajournals.org.