Aortic Valve Stenosis Causes Accumulation of Extracellular Hemoglobin and Systemic Endothelial Dysfunction.

BACKGROUND: Whether aortic valve stenosis (AS) can adversely affect systemic endothelial function independently of standard modifiable cardiovascular risk factors is unknown. METHODS: We therefore investigated endothelial and cardiac function in an experimental model of AS mice devoid of standard modifiable cardiovascular risk factors and human cohorts with AS scheduled for transcatheter aortic valve replacement. Endothelial function was determined by flow-mediated dilation using ultrasound. Extracellular hemoglobin (eHb) concentrations and nitric oxide (NO) consumption were determined in blood plasma of mice and humans by ELISA and chemiluminescence. This was complemented by measurements of aortic blood flow using 4-dimensional flow acquisition by magnetic resonance imaging and computational fluid dynamics simulations. The effects of plasma and red blood cell (RBC) suspensions on vascular function were determined in transfer experiments in a murine vasorelaxation bioassay system. RESULTS: In mice, the induction of AS caused systemic endothelial dysfunction. In the presence of normal systolic left ventricular function and mild hypertrophy, the increase in the transvalvular gradient was associated with elevated eryptosis, increased eHb, and increased plasma NO consumption; eHb sequestration by haptoglobin restored endothelial function. Because the aortic valve orifice area in patients with AS decreased, postvalvular mechanical stress in the central ascending aorta increased. This was associated with elevated eHb, circulating RBC-derived microvesicles, eryptotic cells, lower haptoglobin levels without clinically relevant anemia, and consecutive endothelial dysfunction. Transfer experiments demonstrated that reduction of eHb by treatment with haptoglobin or elimination of fluid dynamic stress by transcatheter aortic valve replacement restored endothelial function. In patients with AS and subclinical RBC fragmentation, the remaining circulating RBCs before and after transcatheter aortic valve replacement exhibited intact membrane function, deformability, and resistance to osmotic and hypoxic stress. CONCLUSIONS: AS increases postvalvular swirling blood flow in the central ascending aorta, triggering RBC fragmentation with the accumulation of hemoglobin in the plasma. This increases NO consumption in blood, thereby limiting vascular NO bioavailability. Thus, AS itself promotes systemic endothelial dysfunction independent of other established risk factors. Transcatheter aortic valve replacement is capable of limiting NO scavenging and rescuing endothelial function by realigning postvalvular blood flow to near physiological patterns. REGISTRATION: URL: https://www.clinicaltrials.gov; Unique identifiers: NCT05603520 and NCT01805739.

Trained Innate Immunity in Animal Models of Cardiovascular Diseases.

Acquisition of immunological memory is an important evolutionary strategy that evolved to protect the host from repetitive challenges from infectious agents. It was believed for a long time that memory formation exclusively occurs in the adaptive part of the immune system with the formation of highly specific memory T cells and B cells. In the past 10-15 years, it has become clear that innate immune cells, such as monocytes, natural killer cells, or neutrophil granulocytes, also have the ability to generate some kind of memory. After the exposure of innate immune cells to certain stimuli, these cells develop an enhanced secondary response with increased cytokine secretion even after an encounter with an unrelated stimulus. This phenomenon has been termed trained innate immunity (TI) and is associated with epigenetic modifications (histone methylation, acetylation) and metabolic alterations (elevated glycolysis, lactate production). TI has been observed in tissue-resident or circulating immune cells but also in bone marrow progenitors. Risk-factors for cardiovascular diseases (CVDs) which are associated with low-grade inflammation, such as hyperglycemia, obesity, or high salt, can also induce TI with a profound impact on the development and progression of CVDs. In this review, we briefly describe basic mechanisms of TI and summarize animal studies which specifically focus on TI in the context of CVDs.

Cardiac injection of USSC boosts remuscularization of the infarcted heart by shaping the T-cell response.

Regenerating the injured heart remains one of the most vexing challenges in cardiovascular medicine. Cell therapy has shown potential for treatment of myocardial infarction, but low cell retention so far has limited its success. Here we show that intramyocardial injection of highly apoptosis-resistant unrestricted somatic stem cells (USSC) into infarcted rat hearts resulted in an unprecedented thickening of the left ventricular wall with cTnT+/BrdU+ cardiomyocytes that was paralleled by progressively restored ejection fraction. USSC induced significant T-cell enrichment in ischemic tissue with enhanced expression of T-cell related cytokines. Inhibition of T-cell activation by anti-CD28 monoclonal antibody, fully abolished the regenerative response which was restored by adoptive T-cell transfer. Secretome analysis of USSC and lineage tracing studies suggest that USSC secrete paracrine factors over an extended period of time which boosts a T-cell driven endogenous regenerative response mainly from adult cardiomyocytes.

Quantitative assessment of angioplasty-induced vascular inflammation with 19F cardiovascular magnetic resonance imaging.

BACKGROUND: Macrophages play a pivotal role in vascular inflammation and predict cardiovascular complications. Fluorine-19 magnetic resonance imaging (19F MRI) with intravenously applied perfluorocarbon allows a background-free direct quantification of macrophage abundance in experimental vascular disease models in mice. Recently, perfluorooctyl bromide-nanoemulsion (PFOB-NE) was applied to effectively image macrophage infiltration in a pig model of myocardial infarction using clinical MRI scanners. In the present proof-of-concept approach, we aimed to non-invasively image monocyte/macrophage infiltration in response to carotid artery angioplasty in pigs using 19F MRI to assess early inflammatory response to mechanical injury. METHODS: In eight minipigs, two different types of vascular injury were conducted: a mild injury employing balloon oversize angioplasty only (BA, n = 4) and a severe injury provoked by BA in combination with endothelial denudation (BA + ECDN, n = 4). PFOB-NE was administered intravenously three days after injury followed by 1H and 19F MRI to assess vascular inflammatory burden at day six. Vascular response to mechanical injury was validated using X-ray angiography, intravascular ultrasound and immunohistology in at least 10 segments per carotid artery. RESULTS: Angioplasty was successfully induced in all eight pigs. Response to injury was characterized by positive remodeling with predominantly adventitial wall thickening and concomitant infiltration of monocytes/macrophages. No severe adverse reactions were observed following PFOB-NE administration. In vivo 19F signals were only detected in the four pigs following BA + ECDN with a robust signal-to-noise ratio (SNR) of 14.7 ± 4.8. Ex vivo analysis revealed a linear correlation of 19F SNR to local monocyte/macrophage cell density. Minimum detection limit of infiltrated monocytes/macrophages was estimated at approximately 410 cells/mm2. CONCLUSIONS: In this proof-of-concept study, 19F MRI enabled quantification of monocyte/macrophage infiltration after vascular injury with sufficient sensitivity. This may provide the opportunity to non-invasively monitor vascular inflammation with MRI in patients after angioplasty or even in atherosclerotic plaques.

SYSTEMI - systemic organ communication in STEMI: design and rationale of a cohort study of patients with ST-segment elevation myocardial infarction.

BACKGROUND: ST-segment elevation myocardial infarction (STEMI) still causes significant mortality and morbidity despite best-practice revascularization and adjunct medical strategies. Within the STEMI population, there is a spectrum of higher and lower risk patients with respect to major adverse cardiovascular and cerebral events (MACCE) or re-hospitalization due to heart failure. Myocardial and systemic metabolic disorders modulate patient risk in STEMI. Systematic cardiocirculatory and metabolic phenotyping to assess the bidirectional interaction of cardiac and systemic metabolism in myocardial ischemia is lacking. METHODS: Systemic organ communication in STEMI (SYSTEMI) is an all-comer open-end prospective study in STEMI patients > 18 years of age to assess the interaction of cardiac and systemic metabolism in STEMI by systematically collecting data on a regional and systemic level. Primary endpoint will be myocardial function, left ventricular remodelling, myocardial texture and coronary patency at 6 month after STEMI. Secondary endpoint will be all-cause death, MACCE, and re-hospitalisation due to heart failure or revascularisation assessed 12 month after STEMI. The objective of SYSTEMI is to identify metabolic systemic and myocardial master switches that determine primary and secondary endpoints. In SYSTEMI 150-200 patients are expected to be recruited per year. Patient data will be collected at the index event, within 24 h, 5 days as well as 6 and 12 months after STEMI. Data acquisition will be performed in multilayer approaches. Myocardial function will be assessed by using serial cardiac imaging with cineventriculography, echocardiography and cardiovascular magnetic resonance. Myocardial metabolism will be analysed by multi-nuclei magnetic resonance spectroscopy. Systemic metabolism will be approached by serial liquid biopsies and analysed with respect to glucose and lipid metabolism as well as oxygen transport. In summary, SYSTEMI enables a comprehensive data analysis on the levels of organ structure and function alongside hemodynamic, genomic and transcriptomic information to assess cardiac and systemic metabolism. DISCUSSION: SYSTEMI aims to identify novel metabolic patterns and master-switches in the interaction of cardiac and systemic metabolism to improve diagnostic and therapeutic algorithms in myocardial ischemia for patient-risk assessment and tailored therapy. TRIAL REGISTRATION: Trial Registration Number: NCT03539133.

Diabetes disturbs functional adaptation of the remote myocardium after ischemia/reperfusion.

Diabetes mellitus type 2 is associated with adverse clinical outcome after myocardial infarction. To better understand the underlying causes we here investigated sarcomere protein function and its calcium-dependent regulation in the non-ischemic remote myocardium (RM) of diabetic mice (db/db) after transient occlusion of the left anterior descending coronary artery. Before and 24 h after surgery db/db and non-diabetic db/+ underwent magnetic resonance imaging followed by histological and biochemical analyses of heart tissue. Intracellular calcium transients and sarcomere function were measured in isolated cardiomyocytes. Active and passive force generation was assessed in skinned fibers and papillary muscle preparations. Before ischemia and reperfusion (I/R), beat-to-beat calcium cycling was depressed in diabetic cardiomyocytes. Nevertheless, contractile function was preserved owing to increased myofilament calcium sensitivity and higher responsiveness of myocardial force production to ß-adrenergic stimulation in db/db compared to db/+. In addition, protein kinase C activity was elevated in db/db hearts leading to strong phosphorylation of the titin PEVK region and increased titin-based tension of myofilaments. I/R impaired the function of whole hearts and RM sarcomeres in db/db to a larger extent than in non-diabetic db/+, and we identified several reasons. First, the amplitude and the kinetics of cardiomyocyte calcium transients were further reduced in the RM of db/db. Underlying causes involved altered expression of calcium regulatory proteins. Diabetes and I/R additively reduced phospholamban S16-phosphorylation by 80% (P < 000.1) leading to strong inhibition of the calcium ATPase SERCA2a. Second, titin stiffening was only observed in the RM of db/+, but not in the RM of db/db. Finally, db/db myofilament calcium sensitivity and force generation upon ß-adrenergic stimulation were no longer enhanced over db/+ in the RM. The findings demonstrate that impaired cardiomyocyte calcium cycling of db/db hearts is compensated by increased myofilament calcium sensitivity and increased titin-based stiffness prior to I/R. In contrast, sarcomere function of the RM 24 h after I/R is poor because both these compensatory mechanisms fail and myocyte calcium handling is further depressed.

Multiparametric MRI identifies subtle adaptations for demarcation of disease transition in murine aortic valve stenosis.

Aortic valve stenosis (AS) is the most frequent valve disease with relevant prognostic impact. Experimental model systems for AS are scarce and comprehensive imaging techniques to simultaneously quantify function and morphology in disease progression are lacking. Therefore, we refined an acute murine AS model to closely mimic human disease characteristics and developed a high-resolution magnetic resonance imaging (MRI) approach for simultaneous in-depth analysis of valvular, myocardial as well as aortic morphology/pathophysiology to identify early changes in tissue texture and critical transition points in the adaptive process to AS. AS was induced by wire injury of the aortic valve. Four weeks after surgery, cine loops, velocity, and relaxometry maps were acquired at 9.4 T to monitor structural/functional alterations in valve, aorta, and left ventricle (LV). In vivo MRI data were subsequently validated by histology and compared to echocardiography. AS mice exhibited impaired valve opening accompanied by significant valve thickening due to fibrotic remodelling. While control mice showed bell-shaped flow profiles, AS resulted not only in higher peak flow velocities, but also in fragmented turbulent flow patterns associated with enhanced circumferential strain and an increase in wall thickness of the aortic root. AS mice presented with a mild hypertrophy but unaffected global LV function. Cardiac MR relaxometry revealed reduced values for both T1 and T2 in AS reflecting subtle myocardial tissue remodelling with early alterations in mitochondrial function in response to the enhanced afterload. Concomitantly, incipient impairments of coronary flow reserve and myocardial tissue integrity get apparent accompanied by early troponin release. With this, we identified a premature transition point with still compensated cardiac function but beginning textural changes. This will allow interventional studies to explore early disease pathophysiology and novel therapeutic targets.

Endothelial Hyaluronan Synthase 3 Augments Postischemic Arteriogenesis Through CD44/eNOS Signaling.

Objective: The dominant driver of arteriogenesis is elevated shear stress sensed by the endothelial glycocalyx thereby promoting arterial outward remodeling. Hyaluronan, a critical component of the endothelial glycocalyx, is synthesized by 3 HAS isoenzymes (hyaluronan synthases 1-3) at the plasma membrane. Considering further the importance of HAS3 for smooth muscle cell and immune cell functions we aimed to evaluate its role in collateral artery growth. Approach and Results: Male Has3-deficient (Has3-KO) mice were subjected to hindlimb ischemia. Blood perfusion was monitored by laser Doppler perfusion imaging and endothelial function was assessed by measurement of flow-mediated dilation in vivo. Collateral remodeling was monitored by high resolution magnetic resonance angiography. A neutralizing antibody against CD44 (clone KM201) was injected intraperitoneally to analyze hyaluronan signaling in vivo. After hindlimb ischemia, Has3-KO mice showed a reduced arteriogenic response with decreased collateral remodeling and impaired perfusion recovery. While postischemic leukocyte infiltration was unaffected, a diminished flow-mediated dilation pointed towards an impaired endothelial cell function. Indeed, endothelial AKT (protein kinase B)-dependent eNOS (endothelial nitric oxide synthase) phosphorylation at Ser1177 was substantially reduced in Has3-KO thigh muscles. Endothelial-specific Has3-KO mice mimicked the hindlimb ischemia-induced phenotype of impaired perfusion recovery as observed in global Has3-deficiency. Mechanistically, blocking selectively the hyaluronan binding site of CD44 reduced flow-mediated dilation, thereby suggesting hyaluronan signaling through CD44 as the underlying signaling pathway. Conclusions: In summary, HAS3 contributes to arteriogenesis in hindlimb ischemia by hyaluronan/CD44-mediated stimulation of eNOS phosphorylation at Ser1177. Thus, strategies augmenting endothelial HAS3 or CD44 could be envisioned to enhance vascularization under pathological conditions.

Dapagliflozin reduces thrombin generation and platelet activation: implications for cardiovascular risk reduction in type 2 diabetes mellitus.

AIMS/HYPOTHESIS: People with diabetes have an increased cardiovascular risk with an accelerated development of atherosclerosis and an elevated mortality rate after myocardial infarction. Therefore, cardioprotective effects of glucose-lowering therapies are of major importance for the pharmacotherapy of individuals with type 2 diabetes. For sodium-glucose cotransporter 2 inhibitors (SGLT2is), in addition to a reduction in blood glucose, beneficial effects on atherosclerosis, obesity, renal function and blood pressure have been observed. Recent results showed a reduced risk of worsening heart failure and cardiovascular deaths under dapagliflozin treatment irrespective of the diabetic state. However, the underlying mechanisms are yet unknown. Platelets are known drivers of atherosclerosis and atherothrombosis and disturbed platelet activation has also been suggested to occur in type 2 diabetes. Therefore, the present study investigates the impact of the SGLT2i dapagliflozin on the interplay between platelets and inflammation in atherogenesis. METHODS: Male, 8-week-old LDL-receptor-deficient (Ldlr-/-) mice received a high-fat, high-sucrose diabetogenic diet supplemented without (control) or with dapagliflozin (5 mg/kg body weight per day) for two time periods: 8 and 25 weeks. In a first translational approach, eight healthy volunteers received 10 mg dapagliflozin/day for 4 weeks. RESULTS: Dapagliflozin treatment ameliorated atherosclerotic lesion development, reduced circulating platelet-leucocyte aggregates (glycoprotein [GP]Ib+CD45+: 29.40 ± 5.94 vs 17.00 ± 5.69 cells, p < 0.01; GPIb+lymphocyte antigen 6 complex, locus G+ (Ly6G): 8.00 ± 2.45 vs 4.33 ± 1.75 cells, p < 0.05) and decreased aortic macrophage infiltration (1.31 ± 0.62 vs 0.70 ± 0.58 ×103 cells/aorta, p < 0.01). Deeper analysis revealed that dapagliflozin decreased activated CD62P-positive platelets in Ldlr-/- mice fed a diabetogenic diet (3.78 ± 1.20% vs 2.83 ± 1.06%, p < 0.01) without affecting bleeding time (85.29 ± 37.27 vs 89.25 ± 16.26 s, p = 0.78). While blood glucose was only moderately affected, dapagliflozin further reduced endogenous thrombin generation (581.4 ± 194.6 nmol/l × min) × 10-9 thrombin vs 254.1 ± 106.4 (nmol/l × min) × 10-9 thrombin), thereby decreasing one of the most important platelet activators. We observed a direct inhibitory effect of dapagliflozin on isolated platelets. In addition, dapagliflozin increased HDL-cholesterol levels. Importantly, higher HDL-cholesterol levels (1.70 ± 0.58 vs 3.15 ± 1.67 mmol/l, p < 0.01) likely contribute to dapagliflozin-mediated inhibition of platelet activation and thrombin generation. Accordingly, in line with the results in mice, treatment with dapagliflozin lowered CD62P-positive platelet counts in humans after stimulation by collagen-related peptide (CRP; 88.13 ± 5.37% of platelets vs 77.59 ± 10.70%, p < 0.05) or thrombin receptor activator peptide-6 (TRAP-6; 44.23 ± 15.54% vs 28.96 ± 11.41%, p < 0.01) without affecting haemostasis. CONCLUSIONS/INTERPRETATION: We demonstrate that dapagliflozin-mediated atheroprotection in mice is driven by elevated HDL-cholesterol and ameliorated thrombin-platelet-mediated inflammation without interfering with haemostasis. This glucose-independent mechanism likely contributes to dapagliflozin's beneficial cardiovascular risk profile.

4-hydroxytamoxifen does not deteriorate cardiac function in cardiomyocyte-specific MerCreMer transgenic mice.

Conditional, cell-type-specific transgenic mouse lines are of high value in cardiovascular research. A standard tool for cardiomyocyte-restricted DNA editing is the αMHC-MerCreMer/loxP system. However, there is an ongoing debate on the occurrence of cardiac side effects caused by unspecific Cre activity or related to tamoxifen/oil overload. Here, we investigated potential adverse effects of DNA editing by the αMHC-MerCreMer/loxP system in combination with a low-dose treatment protocol with the tamoxifen metabolite 4-hydroxytamoxifen (OH-Txf). αMHC-MerCreMer mice received intraperitoneally OH-Txf (20 mg/kg) for 5 or 10 days. These treatment protocols were highly efficient to induce DNA editing in adult mouse hearts. Multi-parametric magnetic resonance imaging revealed neither transient nor permanent effects on cardiac function during or up to 19 days after 5 day OH-Txf treatment. Furthermore, OH-Txf did not affect cardiac phosphocreatine/ATP ratios assessed by in vivo 31P MR spectroscopy, indicating no Cre-mediated side effects on cardiac energy status. No MRI-based indication for the development of cardiac fibrosis was found as mean T1 relaxation time was unchanged. Histological analysis of myocardial collagen III content after OH-Txf confirmed this result. Last, mean T2 relaxation time was not altered after Txf treatment suggesting no pronounced cardiac lipid accumulation or tissue oedema. In additional experiments, cardiac function was assessed for up to 42 days to investigate potential delayed side effects of OH-Txf treatment. Neither 5- nor 10-day treatment resulted in a depression of cardiac function. Efficient cardiomyocyte-restricted DNA editing that is free of unwanted side effects on cardiac function, energetics or fibrosis can be achieved in adult mice when the αMHC-MerCreMer/loxP system is activated by the tamoxifen metabolite OH-Txf.

Cardiac Hyaluronan Synthesis Is Critically Involved in the Cardiac Macrophage Response and Promotes Healing After Ischemia Reperfusion Injury.

RATIONALE: Immediate changes in the ECM (extracellular matrix) microenvironment occur after myocardial ischemia and reperfusion (I/R) injury. OBJECTIVE: Aim of this study was to unravel the role of the early hyaluronan (HA)-rich ECM after I/R. METHODS AND RESULTS: Genetic deletion of Has2 and Has1 was used in a murine model of cardiac I/R. Chemical exchange saturation transfer imaging was adapted to image cardiac ECM post-I/R. Of note, the cardiac chemical exchange saturation transfer signal was severely suppressed by Has2 deletion and pharmacological inhibition of HA synthesis 24 hours after I/R. Has2 KO ( Has2 deficient) mice showed impaired hemodynamic function suggesting a protective role for endogenous HA synthesis. In contrast to Has2 deficiency, Has1-deficient mice developed no specific phenotype compared with control post-I/R. Importantly, in Has2 KO mice, cardiac macrophages were diminished after I/R as detected by 19F MRI (magnetic resonance imaging) of perfluorcarbon-labeled immune cells, Mac-2/Galectin-3 immunostaining, and FACS (fluorescence-activated cell sorting) analysis (CD45+CD11b+Ly6G-CD64+F4/80+cells). In contrast to macrophages, cardiac Ly6Chigh and Ly6Clow monocytes were unaffected post-I/R compared with control mice. Mechanistically, inhibition of HA synthesis led to increased macrophage apoptosis in vivo and in vitro. In addition, α-SMA (α-smooth muscle actin)-positive cells were reduced in the infarcted myocardium and in the border zone. In vitro, the myofibroblast response as measured by Acta2 mRNA expression was reduced by inhibition of HA synthesis and of CD44 signaling. Furthermore, Has2 KO fibroblasts were less able to contract collagen gels in vitro. The effects of HA/CD44 on fibroblasts and macrophages post-I/R might also affect intercellular cross talk because cardiac fibroblasts were activated by monocyte/macrophages and, in turn, protected macrophages from apoptosis. CONCLUSIONS: Increased HA synthesis contributes to postinfarct healing by supporting macrophage survival and by promoting the myofibroblast response. Additionally, imaging of cardiac HA by chemical exchange saturation transfer post-I/R might have translational value.

Anaemia is associated with severe RBC dysfunction and a reduced circulating NO pool: vascular and cardiac eNOS are crucial for the adaptation to anaemia.

Anaemia is frequently present in patients with acute myocardial infarction (AMI) and contributes to an adverse prognosis. We hypothesised that, besides reduced oxygen carrying capacity, anaemia is associated with (1) red blood cell (RBC) dysfunction and a reduced circulating nitric oxide (NO) pool, (2) compensatory enhancement of vascular and cardiac endothelial nitric oxide synthase (eNOS) activity, and (3) contribution of both, RBC dysfunction and reduced circulatory NO pool to left ventricular (LV) dysfunction and fatal outcome in AMI. In mouse models of subacute and chronic anaemia from repeated mild blood loss the circulating NO pool, RBC, cardiac and vascular function were analysed at baseline and in reperfused AMI. In anaemia, RBC function resulted in profound changes in membrane properties, enhanced turnover, haemolysis, dysregulation of intra-erythrocytotic redox state, and RBC-eNOS. RBC from anaemic mice and from anaemic patients with acute coronary syndrome impaired the recovery of contractile function of isolated mouse hearts following ischaemia/reperfusion. In anaemia, the circulating NO pool was reduced. The cardiac and vascular adaptation to anaemia was characterised by increased arterial eNOS expression and activity and an eNOS-dependent increase of end-diastolic left ventricular volume. Endothelial dysfunction induced through genetic or pharmacologic reduction of eNOS-activity abrogated the anaemia-induced cardio-circulatory compensation. Superimposed AMI was associated with decreased survival. In summary, moderate blood loss anaemia is associated with severe RBC dysfunction and reduced circulating NO pool. Vascular and cardiac eNOS are crucial for the cardio-circulatory adaptation to anaemia. RBC dysfunction together with eNOS dysfunction may contribute to adverse outcomes in AMI.

A2bR-dependent signaling alters immune cell composition and enhances IL-6 formation in the ischemic heart.

Although the cardioprotective effect of adenosine is undisputed, the role of the adenosine A2b receptor (A2bR) in ischemic cardiac remodeling is not defined. In this study we aimed to unravel the role A2bR plays in modulating the immune response and the healing mechanisms after myocardial infarction. Genetic and pharmacological (PSB603) inactivation of A2bR as well as activation of A2bR with BAY60-6583 does not alter cardiac remodeling of the infarcted (50-min left anterior descending artery occlusion/reperfusion) murine heart. Flow cytometry of immune cell subsets identified a significant increase in B cells, NK cells, CD8 and CD4 T cells, as well as FoxP3-expressing regulatory T cells in the injured heart in A2bR-deficient mice. Analysis of T-cell function revealed that expression and secretion of interleukin (IL)-2, interferon (IFN)γ, and tumor necrosis factor (TNF)α by T cells is under A2bR control. In addition, we found substantial cellular heterogeneity in the response of immune cells and cardiomyocytes to A2bR deficiency: while in the absence of A2bR, expression of IL-6 was greatly reduced in cardiomyocytes and immune cells except T cells, and expression of IL-1ß was strongly reduced in cardiomyocytes, granulocytes, and B cells as determined by quantitative PCR. Our findings indicate that A2bR signaling in the ischemic heart triggers substantial changes in cardiac immune cell composition of the lymphoid lineage and induces a profound cell type-specific downregulation of IL-6 and IL-1ß. This suggests the presence of a targetable adenosine-A2bR-IL-6-axis triggered by adenosine formed by the ischemic heart. NEW & NOTEWORTHY Genetic deletion and pharmacological inactivation/activation of A2bR does not alter cardiac remodeling after MI but is associated by compensatory upregulation of various pro- and anti-inflammatory immune cell subsets (B cells, NK cells, CD8 and CD4 T cells, regulatory T cells). In the inflamed heart, A2bR modulates the expression of IL-2, IFNγ, TNFα in T cells and of IL-6 in cardiomyocytes, monocytes, granulocytes and B cells. This suggests an important adenosine-IL-6 axis, which is controlled by A2bR via local adenosine.

Dissociation of 19F and fluorescence signal upon cellular uptake of dual-contrast perfluorocarbon nanoemulsions.

OBJECTIVE: Perfluorocarbon nanoemulsions (PFCs) tagged with fluorescence dyes have been intensively used to confirm the in vivo 19F magnetic resonance imaging (MRI) localization of PFCs by post mortem histology or flow cytometry. However, only limited data are available on tagged PFCs and the potential dissociation of fluorescence and 19F label after cellular uptake over time. MATERIALS AND METHODS: PFCs were coupled to rhodamine (Rho) or carboxyfluorescein (Cfl) and their fate was analyzed after in vitro uptake by J774, RAW and CHO cells by flow cytometry and 19F MRI. In separate in vivo experiments, the dual-labelled emulsions were intravenously applied into mice and their distribution was monitored in spleen and liver over 24 h. In a final step, time course of fluorescence and 19F signals from injected emulsions were tracked in a local inflammation model making use of a subcutaneous matrigel depot doped with LPS (lipopolysaccharide). RESULTS: Internalization of fluorescence-labelled PFCs was associated with a substantial whitening over 24 h in all macrophage cell lines while the 19F signal remained stable over time. In all experiments, CflPFCs were more susceptible to bleaching than RhoPFCs. After intravenous injection of RhoPFCs, the fluorescence signal in spleen and liver peaked after 30 min and 2 h, respectively, followed by a successive decrease over 24 h, whereas the 19F signal continuously increased during this observation period. Similar results were found in the matrigel/LPS model, where we observed increasing 19F signals in the inflammatory hot spot over time while the fluorescence signal of immune cells isolated from the matrigel depot 24 h after its implantation was only marginally elevated over background levels. This resulted in a massive underestimation of the true PFC deposition in the reticuloendothelial system and at inflammatory hot spots. CONCLUSION: Cellular uptake of fluorescently tagged PFCs leads to a dissociation of the fluorescence and the 19F label signal over time, which critically impacts on interpretation of long-term experiments validated by histology or flow cytometry.

In vivo 19F MR inflammation imaging after myocardial infarction in a large animal model at 3 T.

OBJECTIVES: Fluorine-19 (19F) MRI with intravenously applied perfluorocarbons allows the in vivo monitoring of infiltrating immune cells as demonstrated in small animal models at high field. Here, we aimed to transfer this approach to a clinical scanner for detection of inflammatory processes in the heart after acute myocardial infarction (AMI) in a large animal model. MATERIALS AND METHODS: Optimization of coil and sequence performance was carried out on phantoms and in vivo at a 3 T Philips Achieva. AMI was induced in Munich mini pigs by 90-min occlusion of the left anterior descending artery. At day 3 after AMI, pigs received a body weight-adjusted intravenous dose of a perfluorooctyl bromide nanoemulsion followed by 1H/19F MRI at day 6 after AMI. RESULTS: A balanced steady-state free precession turbo gradient echo sequence using an ellipsoidal 19F/1H surface coil provided the best signal-to-noise ratio and a superior localization of 19F patterns in vivo. This approach allowed the reliable detection of 19F signals in the injured myocardium within less than 20 min. The 19F signal magnitude correlated significantly with the functional impairment after AMI. CONCLUSION: This study demonstrates the feasibility of in vivo 19F MR inflammation imaging after AMI at 3 T within a clinically acceptable acquisition time.

Longitudinal 19F magnetic resonance imaging of brain oxygenation in a mouse model of vascular cognitive impairment using a cryogenic radiofrequency coil.

INTRODUCTION: We explored the use of a perfluoro-15-crown-5 ether nanoemulsion (PFC) for measuring tissue oxygenation using a mouse model of vascular cognitive impairment. METHODS: Seventeen C57BL/6 mice underwent stereotactic injection of PFC coupled to a fluorophore into the striatum and corpus callosum. Combined 1H/19F magnetic resonance imaging (MRI) to localize the PFC and R1 mapping to assess pO2 were performed. The effect of gas challenges on measured R1 was investigated. All mice then underwent bilateral implantation of microcoils around the common carotid arteries to induce global cerebral hypoperfusion. 19F-MRI and R1 mapping were performed 1 day, 1 week, and 4 weeks after microcoil implantation. In vivo R1 values were converted to pO2 through in vitro calibration. Tissue reaction to the PFC was assessed through ex vivo immunohistochemistry of microglial infiltration. RESULTS: R1 increased with increasing oxygen concentrations both in vitro and in vivo and the strength of the 19F signal remained largely stable over 4 weeks. In the two mice that received all four scans, tissue pO2 decreased after microcoil implantation and recovered 4 weeks later. We observed infiltration of the PFC deposits by microglia. DISCUSSION: Despite remaining technical challenges, intracerebrally injected PFC is suitable for monitoring brain oxygenation in vivo.