Zhaoping Ding

Coronary Hemodynamics in Endothelial NO Synthase Knockout Mice

For the specific analysis of endothelial NO synthase (eNOS) function in the coronary vasculature, we generated a mouse homozygous for a defective eNOS gene (eNOS-/-). Western blot as well as immunohistochemical staining revealed the absence of eNOS protein in eNOS-/- mice. Aortic endothelial cells derived from eNOS-/- mice displayed only background levels of NOx formation compared with wild-type (WT) cells (88 versus 1990 pmol NOx x h-1/mg protein-1). eNOS-/- mice were hypertensive (mean arterial pressure, 135 +/- 15 versus 107 +/- 8 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 Nomega-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, -35%) in WT hearts was followed by sustained vasodilation (+190%). L-NAME significantly reduced vasodilation in WT hearts (+125%) but did not alter the initial vasoconstriction. In eNOS-/- hearts, the initial vasoconstriction was augmented (-70%), whereas the ACh-induced vasodilation was not affected. Inhibition of cyclooxygenase with diclofenac converted the ACh-induced vasodilation into vasoconstriction (-49% decrease of basal flow). This effect was even more pronounced in eNOS-/- hearts (-71%). 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 eNOS-/- mutant mice induces no changes in basal coronary flow and reactive hyperemia, suggesting the activation of important compensatory mechanisms; and (3) prostaglandins are the main mediators of the ACh-induced vasodilation in both WT and eNOS-/- mice.

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.

Targeted Disruption of cd73/Ecto-5′-Nucleotidase Alters Thromboregulation and Augments Vascular Inflammatory Response

To investigate the role of adenosine formed extracellularly in vascular homeostasis, mice with a targeted deletion of the cd73/ecto-5′-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 (−14%; P<0.05) 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 (FeCl3) was significantly reduced by 20% in cd73−/− mice (P<0.05). Bleeding time after tail tip resection tended to be shorter in cd73−/− mice (−35%). In vivo platelet cAMP levels were 0.96±0.46 in WT versus 0.68±0.27 pmol/106 cells in cd73−/− mice (P<0.05). Under in vitro conditions, platelet aggregation in response to ADP (0.05 to 10 &mgr;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−/− compared with WT littermates (WT 98% versus cd73−/− 245%; P<0.005). The constitutive adhesion of monocytes in ex vivo–perfused carotid arteries of WT mice was negligible but significantly increased in arteries of cd73−/− mice (P<0.05). 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.

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.

Deficiency of Biglycan Causes Cardiac Fibroblasts to Differentiate into a Myofibroblast Phenotype

Myocardial infarction (MI) is followed by extracellular matrix (ECM) remodeling, which is on the one hand required for the healing response and the formation of stable scar tissue. However, on the other hand, ECM remodeling can lead to fibrosis and decreased ventricular compliance. The small leucine-rich proteoglycan (SLRP), biglycan (bgn), has been shown to be critically involved in these processes. During post-infarct remodeling cardiac fibroblasts differentiate into myofibroblasts which are the main cell type mediating ECM remodeling. The aim of the present study was to characterize the role of bgn in modulating the phenotype of cardiac fibroblasts. Cardiac fibroblasts were isolated from hearts of wild-type (WT) versus bgn−/0 mice. Phenotypic characterization of the bgn−/0 fibroblasts revealed increased proliferation. Importantly, this phenotype of bgn−/0 fibroblasts was abolished to the WT level by reconstitution of biglycan in the ECM. TGF-β receptor II expression and phosphorylation of SMAD2 were increased. Furthermore, indicative of a myofibroblast phenotype bgn−/0 fibroblasts were characterized by increased α-smooth muscle actin (α-SMA) incorporated into stress fibers, increased formation of focal adhesions, and increased contraction of collagen gels. Administration of neutralizing antibodies to TGF-β reversed the pro-proliferative, myofibroblastic phenotype. In vivo post-MI α-SMA, TGF-β receptor II expression, and SMAD2 phosphorylation were markedly increased in bgn−/0 mice. Collectively, the data suggest that bgn deficiency promotes myofibroblast differentiation and proliferation in vitro and in vivo likely due to increased responses to TGF-β and SMAD2 signaling.

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.

Noninvasive Detection of Graft Rejection by In Vivo 19F MRI in the Early Stage

Diagnosis of transplant rejection requires tissue biopsy and entails risks. Here, we describe a new 19F MRI approach for noninvasive visualization of organ rejection via the macrophage host response. For this, we employed biochemically inert emulsified perfluorocarbons (PFCs), known to be preferentially phagocytized by monocytes and macrophages. Isografts from C57BL/6 or allografts from C57B10.A mice were heterotopically transplanted into C57BL/6 recipients. PFCs were applied intravenously followed by 1H/19F MRI at 9.4 T 24 h after injection. 1H images showed a similar position and anatomy of the graft in the abdomen for both cases. However, corresponding 19F signals were only observed in allogenic tissue. 1H/19F MRI enabled us to detect the initial immune response not later than 3 days after surgery, when conventional parameters did not reveal any signs of rejection. In allografts, the observed 19F signal strongly increased with time and correlated with the extent of rejection. In separate experiments, rapamycin was used to demonstrate the ability of 19F MRI to monitor immunosuppressive therapy. Thus, PFCs can serve as positive contrast agent for the early detection of transplant rejection by 19F MRI with high spatial resolution and an excellent degree of specificity due to lack of any 19F background.

Endothelial dysfunction of coronary resistance vessels in apoE-/-mice involves NO but not prostacyclin-dependent mechanisms

OBJECTIVE We have analyzed the extent of endothelial dysfunction in cardiac resistance vessels of hyperlipidaemic apoE-/- mice and explored whether NO and/or prostacyclin dependent pathways are involved. METHODS Coronary resistance was measured in isolated perfused hearts from WT and apoE-/- mice. To discriminate between NO and PGI(2)-dependent flow responses, we made use of the finding that acetylcholine (ACh) predominantly activates the prostaglandin pathway whereas bradykinin (Bk) mainly acts via NO in murine cardiac resistance vessels. RESULTS Basal coronary flow as well as the ACh induced vasodilation (0.1-1 microM) were not different between WT and apoE-/- hearts (flow increase+100%). Similarly, vasodilation in response to the prostacyclin mimetic iloprost reached the same levels. In contrast, the Bk-stimulated [3.3 microM Bk] coronary flow was reduced from 31.6+/-4.2 in WT to 19.2+/-2.7 ml min(-1) g(-1) in apoE-/- hearts. NOS inhibition by ethylisothiourea (ETU, 10 microM) reduced basal as well as Bk-stimulated coronary flow in WT and apoE-/- hearts to the same extent. RT-PCR and Western analysis demonstrated that neither eNOS expression nor protein levels were reduced. Similarly, the flow response to the NO donor SNAP (0.3-33 microM) was not altered suggesting that soluble guanylyl cyclase was not affected. Intracoronary application of superoxide dismutase augmented the Bk-induced vasodilation of apoE-/- hearts almost back to WT levels (26.6+/-3.3 ml min(-1) g(-1)). In line with this finding the NADPH induced O(2)(-) formation was enhanced in cardiac extracts from apoE-/- hearts. CONCLUSION apoE-/- hearts develop a hemodynamically relevant endothelial dysfunction at the level of coronary resistance vessels most likely via inactivation of bioavailable NO by superoxide anions. The function of the prostacyclin system is not altered.

A minimally invasive approach for efficient gene delivery to rodent hearts.

Transcoronary gene delivery represents a desirable option to achieve global myocardial transgene expression but still requires aggressive surgical preparation in rodents. We therefore developed a catheter-based approach for cardiac gene transfer in the closed chest rat. A double-lumen balloon catheter was used to create aortic occlusion for specific infusion of adenoviral vectors carrying a β-galactosidase transgene (1 × 1011 PFU) into the coronaries. Simultaneously, venous return was obstructed by a second balloon catheter in the right atrium. To prolong viral incubation time, we induced a transient cardiac arrest (2 and 5 min) by a combination of acetylcholine and the β-receptor antagonist, esmolol. At 72 h after transfection, the hearts showed a homogeneous and widespread β-galactosidase expression, and the transduction efficiency increased and up to about 43% of cardiac myocytes (histochemistry) with a 400-fold increase of β-galactosidase activity (luminescence assay) compared to sham-operated hearts. Pharmacological treatment aimed at increasing vascular permeability (SNAP and histamine) did not bring about synergistic effects on transfection efficiency. In addition, the method using high intracoronary pressure delivery (>300 mmHg) in a single-pass manner resulted in rather sparse β-galactosidase expression in the myocardium (3–5% of cardiac myocytes). Therefore, the percutaneous gene delivery system described here provides a simple and minimally invasive procedure that represents a novel strategy for a homogeneous and highly efficient in vivo gene transfer to rodent hearts. Our results also suggest that prolongation of viral incubation time is an effective means for achieving highly efficient myocardial gene transduction.

Nitrosative Stress Leads to Protein Glutathiolation, Increased S-Nitrosation, and Up-regulation of Peroxiredoxins in the Heart

Nitric oxide (NO) is produced by different isoforms of nitric oxide synthases (NOSs) and operates as a mediator of important cell signaling pathways, such as the cGMP signaling cascade. Another mechanism by which NO exerts biological effects is mediated through S-nitrosation of target proteins. To explore thiol-based protein modifications in a situation of defined nitrosative stress, we used a transgenic mouse model with cardiac specific overexpression of inducible nitric oxide synthase (iNOS) and concomitant myoglobin deficiency (iNOS+/myo-/-). In comparison with the wild type hearts, protein glutathiolation detected by immunoblotting was significantly enhanced in iNOS+/myo-/- hearts, whereas protein S-nitrosation as measured by the biotin switch assay and two-dimensional PAGE revealed that nearly all of the detected proteins (∼60) remained unchanged with the exception of three proteins. Tandem mass spectrometry revealed these proteins to be peroxiredoxins (Prxs), which are known to possess peroxidase activity, whereby hydrogen peroxide, peroxynitrite, and a wide range of organic hydroperoxides are reduced and detoxified. Immunoblotting with specific antibodies revealed up-regulation of Prx VI in the iNOS+/myo-/- hearts, whereas expression of Prx II and Prx III remained unchanged. Furthermore, the analysis of the cardiac S-nitrososubproteome identified several new proteins possibly being involved in NO-signaling pathways. Our data indicate that S-nitrosation and glutathiolation of cardiac proteins may contribute to the phenotype of NO-induced heart failure. The up-regulation of antioxidant proteins like Prx VI appears to be an additional mechanism to antagonize an excess of reactive oxygen/nitrogen species. Furthermore, S-nitrosation of Prxs may serve a new function in the signaling cascade of nitrosative stress.