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Benedikt Deuringer,1 Carmen Härdtner,2 Katja Krebs,2 Ralf Thomann,3 Martin Holzer,1 Ingo Hilgendorf,2,4,* Regine Süss1,* 1Department of Pharmaceutical Technology and Biopharmacy, Institute of Pharmaceutical Sciences, University of Freiburg, Freiburg, 79104, Germany; 2Department of Cardiology and Angiology, University Heart Center Freiburg-Bad Krozingen and Faculty of Medicine, University of Freiburg, Freiburg, 79106, Germany; 3FMF Materials Research Center, University of Freiburg, Freiburg, 79104, Germany; 4Institute for Experimental Cardiovascular Medicine, University Heart Center Freiburg-Bad Krozingen and Faculty of Medicine, University of Freiburg, Freiburg, 79110, Germany*These authors contributed equally to this workCorrespondence: Benedikt Deuringer, Pharmaceutical Technology and Biopharmacy, Sonnenstraße 5, Freiburg, 79104, Germany, Tel +49 761 203 6329, Fax +49 761 203 6326, Email bdeuringer@pharmazeutischetechnologie.dePurpose: The conventional techniques for the preparation of reconstituted high-density lipoprotein (rHDL) are hampered by long process times, the need for large amounts of starting material, and harsh preparation conditions. Here, we present a novel rHDL preparation method to overcome these challenges. Furthermore, we propose a dual mode of action for rHDL loaded with the immunosuppressant drug everolimus (Eve-rHDL) in the context of atherosclerosis and cardiovascular disease.Methods: We use dual centrifugation for rHDL nanoparticle preparation and characterize the physicochemical properties by NS-TEM, N-PAGE, DLS, AF4, and HPLC. In addition, we determine the biological efficacy in human and murine cell culture with regard to cellular uptake, cholesterol efflux, and proliferation.Results: We confirm the characteristic particle size of 10 nm, discoidal morphology, and chemical composition of the rHDL preparations and identify dual centrifugation as an ideal method for cost-effective aseptic rHDL manufacturing. rHDL can be prepared in approx. 1.5 h with batch sizes as little as 89 μL. Moreover, we demonstrate the cholesterol efflux capacity and anti-proliferative activity of Eve-rHDL in vitro. The anti-proliferative effects were comparable to free Eve, thus confirming the suitability of rHDL as a capable drug delivery vehicle.Conclusion: Eve-rHDL shows great efficacy in vitro and may further be employed to target atherosclerotic plaques in vivo. Highly effective anti-atherosclerotic therapy might be feasible by reducing both inflammatory- and lipid burden of the plaques. Dual centrifugation is an ideal technique for the efficient application of the rHDL platform in cardiovascular disease and beyond.Keywords: atherosclerosis, cholesterol efflux, drug delivery, everolimus, proliferation, protein-conjugate

BACKGROUND Peripheral, non-neuronal serotonin promotes the recruitment of neutrophils to sites of acute inflammation and tissue damage. Direct effects of serotonin on neutrophil function were shown to be involved. However, the influence of serotonin on the endothelial counterpart is unknown. OBJECTIVES To investigate whether serotonin alters the function of endothelial cells in leukocyte recruitment during acute inflammation. METHODS We used two murine models of acute inflammation: intraperitoneal lipopolysaccharide (LPS) injection and mesenteric ischemia/reperfusion injury (I/R). To study effects of peripheral serotonin, leukocyte recruitment and endothelial adhesion molecule expression were compared in wild type (WT) and tryptophan hydroxylase 1 deficient (Tph1-/- ) mice, which are unable to synthesize peripheral serotonin. RESULTS As expected, neutrophil transmigration into the peritoneal cavity following LPS injection was impaired in Tph1-/- mice. Abdominal blood vessels, however, showed no difference in adhesion molecule expression. In the early reperfusion phase after mesenteric I/R, the number of rolling leukocytes was significantly lower in Tph1-/- compared to WT. In line with the LPS model, endothelial adhesion molecule expression was independent of serotonin. In vitro experiments using human umbilical vein endothelial cells (HUVECs) confirmed that serotonin does not affect endothelial adhesion molecules. CONCLUSIONS The inflammatory release of peripheral serotonin is dispensable for the regulation of endothelial adhesion molecules.

Combined implantation of cardiac contractility modulation (CCM) with subcutaneous implantable cardioverter-defibrillator (S-ICD) appears a suitable option to reduce the amount of intracardiac leads and complications for patients. Here we report on a patient with ischemic cardiomyopathy carrying an S-ICD in which a CCM device was implanted. During crosstalk testing post-CCM implantation, the S-ICD misannotated QRS complexes and T waves. The problem was solved through reprogramming the CCM, while preserving S-ICD functionality and improving heart failure symptoms. In conclusion, S-ICD combined with CCM seems to be a good and safe option for patients when device interference is being ruled out.

Extracellular nucleotides act as danger signals that orchestrate inflammation by purinergic receptor activation. The expression pattern of different purinergic receptors may correlate with a pro- or anti-inflammatory phenotype. Macrophages function as pro-inflammatory M1 macrophages (M1) or anti-inflammatory M2 macrophages (M2). The present study found that murine bone marrow-derived macrophages express a unique purinergic receptor profile during in vitro polarization. As assessed by real-time polymerase chain reaction (PCR), Gαs-coupled P1 receptors A2A and A2B are upregulated in M1 and M2 compared to M0, but A2A 15 times higher in M1. The ionotropic P2 receptor P2X5 is selectively upregulated in M1- and M2-polarized macrophages. P2X7 is temporarily expressed in M1 macrophages. Metabotropic P2Y receptors showed a distinct expression profile in M1 and M2-polarized macrophages: Gαq coupled P2Y1 and P2Y6 are exclusively upregulated in M2, whereas Gαi P2Y13 and P2Y14 are overexpressed in M1. This consequently leads to functional differences between M1 and M2 in response to adenosine di-phosphate stimulation (ADP): In contrast to M1, M2 showed increased cytoplasmatic calcium after ADP stimulation. In the present study we show that bone marrow-derived macrophages express a unique repertoire of purinergic receptors. We show for the first time that the repertoire of purinergic receptors is highly flexible and quickly adapts upon pro- and anti-inflammatory macrophage differentiation with functional consequences to nucleotide stimulation.

Aims P-selectin is an activatable adhesion molecule on platelets promoting platelet aggregation, and platelet–leukocyte complex (PLC) formation. Increased numbers of PLC are circulating in the blood of patients shortly after acute myocardial infarction and predict adverse outcomes. These correlations led to speculations about whether PLC may represent novel therapeutic targets. We therefore set out to elucidate the pathomechanistic relevance of PLC in myocardial ischemia and reperfusion injury. Methods and results By generating P-selectin deficient bone marrow chimeric mice, the post-myocardial infarction surge in PLC numbers in blood was prevented. Yet, intravital microscopy, flow cytometry and immunohistochemical staining, echocardiography, and gene expression profiling showed unequivocally that leukocyte adhesion to the vessel wall, leukocyte infiltration, and myocardial damage post-infarction were not altered in response to the lack in PLC. Conclusion We conclude that myocardial infarction associated sterile inflammation triggers PLC formation, reminiscent of conserved immunothrombotic responses, but without PLC influencing myocardial ischemia and reperfusion injury in return. Our experimental data do not support a therapeutic concept of selectively targeting PLC formation in myocardial infarction.

Aims Macrophages (MΦ), known for immunological roles such as phagocytosis and antigen presentation, have been found to electrotonically couple to cardiomyocytes (CM) of the atrio-ventricular node via Cx43, affecting cardiac conduction in isolated mouse hearts. Here, we characterise passive and active electrophysiological properties of murine cardiac resident MΦ, and model their potential electrophysiological relevance for CM. Methods and results We combined classic electrophysiological approaches with 3 D florescence imaging, RNA-sequencing, pharmacological interventions and computer simulations. We used Cx3cr1eYFP/+ mice wherein cardiac MΦ were fluorescently labelled. FACS-purified fluorescent MΦ from mouse hearts were studied by whole-cell patch-clamp. MΦ electrophysiological properties include: membrane resistance 2.2 ± 0.1 GΩ (all data mean±SEM), capacitance 18.3 ± 0.1 pF, resting membrane potential -39.6 ± 0.3 mV, and several voltage-activated, outward or inwardly-rectifying potassium currents. Using ion channel blockers (barium, TEA, 4-AP, margatoxin, XEN-D0103, DIDS), flow cytometry, immuno-staining and RNA-sequencing, we identified Kv1.3, Kv1.5 and Kir2.1 as channels contributing to observed ion currents. MΦ displayed four patterns for outward and two for inward-rectifier potassium currents. Additionally, MΦ showed surface expression of Cx43, a prerequisite for homo- and/or heterotypic electrotonic coupling. Experimental results fed into development of an original computational model to describe cardiac MΦ electrophysiology. Computer simulations to quantitatively assess plausible effects of MΦ on electrotonically coupled CM showed that MΦ can depolarise resting CM, shorten early and prolong late action potential duration, with effects depending on coupling strength and individual MΦ electrophysiological properties, in particular resting membrane potential and presence/absence of Kir2.1. Conclusions Our results provide a first electrophysiological characterisation of cardiac resident MΦ, and a computational model to quantitatively explore their relevance in the heterocellular heart. Future work will be focussed at distinguishing electrophysiological effects of MΦ-CM coupling on both cell types during steady-state and in patho-physiological remodelling, when immune cells change their phenotype, proliferate, and/or invade from external sources. Translational perspective Cardiac tissue contains resident macrophages (MΦ) which, beyond immunological and housekeeping roles, have been found to electrotonically couple via connexins to cardiomyocytes (CM), stabilising atrio-ventricular conduction at high excitation rates. Here, we characterise structure and electrophysiological function of murine cardiac MΦ and provide a computational model to quantitatively probe the potential relevance of MΦ-CM coupling for cardiac electrophysiology. We find that MΦ are unlikely to have major electrophysiological effects in normal tissue, where they would hasten early and slow late CM-repolarisation. Further work will address potential arrhythmogenicity of MΦ in patho-physiologically remodelled tissue containing elevated MΦ-numbers, incl. non-resident recruited cells.

Background The COVID-19 pandemic led to extensive restrictions in Germany in 2020, including the postponement of elective interventions. We examined the impact on ST-elevation myocardial infarction (STEMI) as an acute and non-postponable disease. Methods Using German national records, all STEMI between 2017 and 2020 were identified. Using the number of STEMI cases between 2017 and 2019, we created a forecast for 2020 and compared it with the observed number of STEMI in 2020. Results From 2017 to 2020, 248,062 patients were treated for STEMI in Germany. Mean age was 65.21 years and 28.36% were female. When comparing forecasted and observed STEMI in 2020, a correlation can be seen: noticeable fewer STEMI were treated in those weeks respectively months with an increasing COVID-19 hospitalization rate (monthly percentage decrease in STEMI: March − 14.85%, April − 13.39%, November − 11.92%, December − 22.95%). At the same time, the crude in-hospital mortality after STEMI increased significantly at the peaks of the first and second waves (relative risk/RR of monthly in-hospital mortality: April RR = 1.11 [95% CI 1.02; 1.21], November RR = 1.13 [1.04; 1.24], December RR = 1.16 [1.06; 1.27]). Conclusion The COVID-19 pandemic led to a noticeable decrease in the number of STEMI interventions in Germany at the peaks of the first and second waves in 2020, corresponding to an increase in COVID-19 hospitalizations. At the same time, in-hospital mortality after STEMI increased significantly in these phases. Graphical abstract Impact of the COVID-19 pandemic on STEMI numbers and in-hospital mortality in Germany. Relative difference between forecasted and observed STEMI numbers (above figure), the relative risk of in-hospital mortality (middle figure) as well as number of new hospital admissions for COVID-19 per million inhabitants according to Roser et al.27 (bottom figure).

Emergency hematopoiesis is the driving force of the inflammatory response to myocardial infarction (MI). Increased proliferation of hematopoietic stem and progenitor cells (LSK) after MI enhances cell production in the bone marrow (BM) and replenishes leukocyte supply for local cell recruitment to the infarct. Decoding the regulation of the inflammatory cascade after MI may provide new avenues to improve post-MI remodeling. In this study, we describe the influence of adenosine diphosphate (ADP)-dependent P2Y12-mediated signaling on emergency hematopoiesis and cardiac remodeling after MI. Permanent coronary ligation was performed to induce MI in a murine model. BM activation, inflammatory cell composition and cardiac function were assessed using global and platelet-specific gene knockout and pharmacological inhibition models for P2Y12. Complementary in vitro studies allowed for investigation of ADP-dependent effects on LSK cells. We found that ADP acts as a danger signal for the hematopoietic BM and fosters emergency hematopoiesis by promoting Akt phosphorylation and cell cycle progression. We were able to detect P2Y12 in LSK, implicating a direct effect of ADP on LSK via P2Y12 signaling. P2Y12 knockout and P2Y12 inhibitor treatment with prasugrel reduced emergency hematopoiesis and the excessive inflammatory response to MI, translating to lower numbers of downstream progeny and inflammatory cells in the blood and infarct. Ultimately, P2Y12 inhibition preserved cardiac function and reduced chronic adverse cardiac remodeling after MI. P2Y12-dependent signaling is involved in emergency hematopoiesis after MI and fuels post-ischemic inflammation, proposing a novel, non-canonical value for P2Y12 antagonists beyond inhibition of platelet-mediated atherothrombosis.

Identifying novel pathways regulating the adaptive immune response in chronic inflammatory diseases such as atherosclerosis is of particular interest in view of developing new therapeutic drugs. Here we report that the lipid receptor GPR55 is highly expressed by splenic B cells and inversely correlates with atheroma plaque size in mice. In human carotid endarterectomy specimen, GPR55 transcript levels were significantly lower in unstable compared to stable carotid plaques. To study the impact of GPR55 deficiency in atherosclerosis, we crossedGpr55knockout mice with apolipoprotein E (ApoE) knockout mice and subjected the mice to Western diet for 4 to 16 weeks. Compared toApoE-/-controls,ApoE-/-Gpr55-/-mice developed larger plaques with increased necrotic core size, associated with elevated circulating and aortic leukocyte counts. Flow cytometry, immunofluorescence and RNA-sequencing analysis of splenic B cells in these mice revealed a hyperactivated B cell phenotype with disturbed plasma cell maturation and immunoglobulin (Ig)G antibody overproduction. The specific contribution of B cell GPR55 in atherosclerosis was further studied in mixedGpr55-/-/µMTbone marrow chimeras on low density receptor deficiency (Ldlr-/-) background, revealing that B-cell specific depletion ofGpr55was sufficient to promote plaque development. Conversely, adoptive transfer of wildtype B cells intoApoE-/-Gpr55-/-mice blunted the proatherogenic phenotype.In vitrostimulation of splenocytes with the endogenous GPR55 ligand LPI promoted plasma cell proliferation and enhanced B cell activation marker expression, which was inhibited by the GPR55 antagonist CID16020046. Collectively, these discoveries provide new evidence for GPR55 as key modulator of the adaptive immune response in atherosclerosis. Targeting GPR55 could be useful to limit inflammation and plaque progression in patients suffering from atherosclerosis.

Objective Impaired cardiac efficiency is a hallmark of diabetic cardiomyopathy in models of type 2 diabetes. Adiponectin receptor 1 (AdipoR1) deficiency impairs cardiac efficiency in non-diabetic mice, suggesting that hypoadiponectinemia in type 2 diabetes may contribute to impaired cardiac efficiency due to compromised AdipoR1 signaling. Thus, we investigated whether targeting cardiac adiponectin receptors may improve cardiac function and energetics, and attenuate diabetic cardiomyopathy in type 2 diabetic mice. Methods A non-selective adiponectin receptor agonist, AdipoRon, and vehicle were injected intraperitoneally into Eight-week-old db/db or C57BLKS/J mice for 10 days. Cardiac morphology and function were evaluated by echocardiography and working heart perfusions. Results Based on echocardiography, AdipoRon treatment did not alter ejection fraction, left ventricular diameters or left ventricular wall thickness in db/db mice compared to vehicle-treated mice. In isolated working hearts, an impairment in cardiac output and efficiency in db/db mice was not improved by AdipoRon. Mitochondrial respiratory capacity, respiration in the presence of oligomycin, and 4-hydroxynonenal levels were similar among all groups. However, AdipoRon induced a marked shift in the substrate oxidation pattern in db/db mice towards increased reliance on glucose utilization. In parallel, the diabetes-associated increase in serum triglyceride levels in vehicle-treated db/db mice was blunted by AdipoRon treatment, while an increase in myocardial triglycerides in vehicle-treated db/db mice was not altered by AdipoRon treatment. Conclusion AdipoRon treatment shifts myocardial substrate preference towards increased glucose utilization, likely by decreasing fatty acid delivery to the heart, but was not sufficient to improve cardiac output and efficiency in db/db mice.

MRI sensitivity for diagnosis and localization of early myocarditis is limited, although it is of central clinical interest. The aim of this project was to test a contrast agent targeting activated platelets consisting of microparticles of iron oxide (MPIO) conjugated to a single-chain antibody directed against ligand-induced binding sites (LIBS) of activated glycoprotein IIb/IIIa (= LIBS-MPIO). Myocarditis was induced by subcutaneous injection of an emulsion of porcine cardiac myosin and complete Freund’s adjuvant in mice. 3D 7 T in-vivo MRI showed focal signal effects in LIBS-MPIO injected mice 2 days after induction of myocarditis, whereas in control-MPIO injected mice no signal was detectable. Histology confirmed CD41-positive staining, indicating platelet involvement in myocarditis in mice as well as in human specimens with significantly higher LIBS-MPIO binding compared to control-MPIO in both species. Quantification of the myocardial MRI signal confirmed a signal decrease after LIBS-MPIO injection and significant less signal in comparison to control-MPIO injection. These data show, that platelets are involved in inflammation during the course of myocarditis in mice and humans. They can be imaged non-invasively with LIBS-MPIO by molecular MRI at an early time point of the inflammation in mice, which is a valuable approach for preclinical models and of interest for both diagnostic and prognostic purposes.

Clonal haematopoiesis of indeterminate potential (CHIP) represents a recently identified overlap between cancer and cardiovascular disease (CVD). CHIP develops as a result of certain acquired somatic mutations that predispose to leukaemia, but clinically even more prevalent, associate with increased risk for CVD and CVD-related death. Experimental studies suggest a causal role for CHIP aggravating inflammatory processes in CVD, and recent epidemiologic and genetic studies indicate that classical CVD risk factors may increase the risk of acquiring CHIP driver mutations, thus fuelling a vicious circle. The potential mechanism underlying the associative link between CHIP and CVD and mortality has been the focus of a few recent excellent experimental and observational studies which are summarized and discussed in this concise non-systematic review article. These data support a pathomechanistic view of a spiralling vicious circle in which CHIP aggravates the inflammatory immune response in CVD, and CVD-driven elevated haematopoietic activity promotes CHIP development.Klonale Hämatopoese von unbestimmtem Potential (CHIP) stellt eine Überschneidung von Krebs- und kardiovaskulären Erkrankungen dar, die erst seit Kurzem Beachtung findet. CHIP entsteht infolge bestimmter erworbener somatischer Mutationen und prädisponiert für die Entwicklung von Leukämien. Aber klinisch häufiger ist die Assoziation mit einem erhöhten Risiko für kardiovaskuläre Erkrankungen und Tod. Experimentelle Studien weisen auf eine kausale Beziehung zwischen CHIP und einem verstärkten Entzündungsprozess bei kardiovaskulären Erkrankungen hin. Epidemiologische und genetische Studien suggerieren umgekehrt, dass die klassischen kardiovaskulären Risikofaktoren das Risiko für die Entwicklung somatischer CHIP-assoziierter Mutationen erhöhen und somit einen Teufelskreis antreiben könnten.

Objectives The co-stimulatory CD40L–CD40 dyad exerts a critical role in atherosclerosis by modulating leukocyte accumulation into developing atherosclerotic plaques. The requirement for cell-type specific expression of both molecules, however, remains elusive. Here, we evaluate the contribution of CD40 expressed on endothelial cells (ECs) in a mouse model of atherosclerosis. Methods and Results Atherosclerotic plaques of apolipoprotein E-deficient (Apoe −/− ) mice and humans displayed increased expression of CD40 on ECs compared with controls. To interrogate the role of CD40 on ECs in atherosclerosis, we induced EC-specific (BmxCreERT2-driven) deficiency of CD40 in Apoe −/− mice. After feeding a chow diet for 25 weeks, EC-specific deletion of CD40 (iEC-CD40) ameliorated plaque lipid deposition and lesional macrophage accumulation but increased intimal smooth muscle cell and collagen content, while atherosclerotic lesion size did not change. Leukocyte adhesion to the vessel wall was impaired in iEC-CD40-deficient mice as demonstrated by intravital microscopy. In accord, expression of vascular cell adhesion molecule 1 (VCAM-1) and intercellular adhesion molecule 1 (ICAM-1) in the vascular endothelium declined after deletion of CD40. In vitro, antibody-mediated inhibition of human endothelial CD40 significantly abated monocyte adhesion on ECs. Conclusion Endothelial deficiency of CD40 in mice promotes structural features associated with a stable plaque phenotype in humans and decreases leukocyte adhesion. These results suggest that endothelial-expressed CD40 contributes to inflammatory cell migration and consecutive plaque formation in atherogenesis.

Previous mouse studies have shown the increased presence of platelets in the myocardium during early stages of myocarditis and their selective detection by MRI. Here, we aimed to depict early myocarditis using molecular contrast-enhanced ultrasound of activated platelets, and to evaluate the impact of a P2Y12 receptor platelet inhibition. Experimental autoimmune myocarditis was induced in BALB/c mice by subcutaneous injection of porcine cardiac myosin and complete Freund adjuvant (CFA). Activated platelets were targeted with microbubbles (MB) coupled to a single-chain antibody that binds to the “ligand-induced binding sites” of the GPIIb/IIIa-receptor (=LIBS-MB). Alongside myocarditis induction, a group of mice received a daily dose of 100 g prasugrel for 1 month. Mice injected with myosin and CFA had a significantly deteriorated ejection fraction and histological inflammation on day 28 compared to mice only injected with myosin. Platelets infiltrated the myocardium before reduction in ejection fraction could be detected by echocardiography. No selective binding of the LIBS-MB contrast agent could be detected by either ultrasound or histology. Prasugrel therapy preserved ejection fraction and significantly reduced platelet aggregates in the myocardium compared to mice without prasugrel therapy. Therefore, P2Y12 inhibition could be a promising early therapeutic target in myocarditis, requiring further investigation.

Extracellular adenosine-5'-triphosphate (ATP) acts as an import signaling molecule mediating inflammation via purinergic P2 receptors. ATP binds to the purinergic receptor P2X

The purpose of this study is to investigate the role of platelet bone morphogenetic proteins (BMP)-4 during vascular inflammation and remodeling in a mouse model of carotid wire injury. Transgenic mice with a platelet-specific deletion of BMP-4 (BMP4Plt−/−) were generated. Intravital microscopy was performed to evaluate leukocyte adhesion to the vessel wall. Expression of adhesion molecules and chemokines were analyzed. Platelet-leukocyte aggregates (PLAs) were evaluated using flow cytometry. For carotid wire injury, BMP4Plt−/− mice were further crossed with LDLr−/− mice (BMP4Plt−/−/LDLr−/−) and fed with a high cholesterol diet for 2-weeks. Carotid wire injury was performed, and re-endothelialization and neointimal formation were evaluated. In comparison to the control mice, stimulation with TNFα resulted in fewer rolling and adherent leukocytes to the vessel wall in the BMP4Plt−/− mice. mRNA and protein expression of P-selectin and adhesion molecules were reduced in the aorta of the BMP4Plt−/− mice. In platelets from the BMP4Plt−/− mice, the expression of P-selectin was reduced, and fewer PLA formations were measured than in the control mice. Loss of platelet BMP-4 further prevented neointima formation after carotid wire injury. Endothelial regeneration after injury was decelerated in the BMP4Plt−/− mice, and confirmed in-vitro, where the deletion of platelet BMP-4 inhibited endothelial cell proliferation and migration. We demonstrate for the first time that platelet BMP-4 is involved during vascular inflammation and remodeling. This is partially mediated by the inhibition of platelet activation, reduced expression of adhesion molecules and inflammatory responses. Our findings identify platelet BMP-4 as a mediator of vascular inflammation in early atherosclerosis and restenosis.

Objectives We aimed to identify risk factors for major transcatheter aortic valve intervention (TAVI) access site complications based on detailed analysis of the preprocedural computed tomography angiogram (CTA). Background Transfemoral TAVI has become the treatment of choice for severe aortic stenosis in elderly patients, especially with increased perioperative risk. Frailty, however, favors complications at the vascular access site due to the large bore vascular sheath devices necessary for valve deployment. Methods In this monocentric study, we retrospectively analyzed the preprocedural CTA of 417 consecutive patients that received transfemoral TAVI between 2015 and 2019 to quantify vessel diameter, calcification volume and calcified plaque location in detail within 10 cm proximal to the femoral bifurcation. Results The mean age of the study cohort was 81.4 ± 6.5 years with a STS of 8 ± 5.2 representing a population at increased periprocedural risk. 54.4% of patients were female. Major vascular access site complications occurred in 8.2% of patients. Major vascular complications correlated statistically with a sheath-to-vessel diameter (SFAR) when measured 1 cm proximal to the femoral bifurcation using a line-derived diameter and ventral calcification within the first 5 cm proximal to the bifurcation. In contrast, overall calcification volume had no influence. Conclusions Transfemoral TAVI harbors a considerable risk for vascular access site complications especially if vessel diameter is too small to comfortably host the sheath diameter at the area of the femoral bifurcation. For preprocedural TAVI planning and risk assessment, location of calcification, especially if located ventrally, seems to be more relevant than consideration of overall calcification alone.

Impaired cardiac efficiency is a hallmark of diabetic cardiomyopathy in models of type 2 diabetes. Adiponectin receptor 1 (AdipoR1) deficiency impairs cardiac efficiency in non-diabetic mice, suggesting that hypoadiponectinemia in type 2 diabetes may contribute to impaired cardiac efficiency due to compromised AdipoR1 signaling. Thus, we investigated whether targeting cardiac adiponectin receptors may improve cardiac function and energetics, and attenuate diabetic cardiomyopathy in type 2 diabetic mice.A non-selective adiponectin receptor agonist, AdipoRon, and vehicle were injected intraperitoneally into Eight-week-oldBased on echocardiography, AdipoRon treatment did not alter ejection fraction, left ventricular diameters or left ventricular wall thickness inAdipoRon treatment shifts myocardial substrate preference towards increased glucose utilization, likely by decreasing fatty acid delivery to the heart, but was not sufficient to improve cardiac output and efficiency in

PURPOSE OF REVIEW Macrophages are key protagonists of atherosclerotic plaque development and hence represent targets of therapeutic intervention. Statins are the most potent widely used atheroprotective drugs. Therefore, whether and how statins influence atheromatous plaque macrophages has remained at the center of cardiovascular research for decades. RECENT FINDINGS Because statins are capable of regulating macrophage functions in cell culture, largely independent of their cholesterol-lowering effect, it was assumed that these pleiotropic effects operate in vivo as well. Recent experimental data, in line with clinical observations, indicate, however, that statins do not interact with macrophages in atherosclerotic plaques, directly, and instead control their functions and assembly indirectly via changes to circulating lipid levels and endothelial activation. SUMMARY Statin-mediated lipid lowering induces plaque regression which is characterized by a decline in plaque macrophage content. Understanding how statins provoke this protective phenotype may inspire conceptually new therapeutic approaches in cardiovascular medicine.

Emergency hematopoiesis is the driving force of the inflammatory response to myocardial infarction (MI). Increased proliferation of hematopoietic stem and progenitor cells (LSK) after MI enhances cell production in the bone marrow (BM) and replenishes leukocyte supply for local cell recruitment to the infarct. Decoding the regulation of the inflammatory cascade after MI may provide new avenues to improve post-MI remodeling. In this study, we describe the influence of adenosine diphosphate (ADP)-dependent P2Y

Similar to the brain, the eye is considered an immune‐privileged organ where tissue‐resident macrophages provide the major immune cell constituents. However, little is known about spatially restricted macrophage subsets within different eye compartments with regard to their origin, function, and fate during health and disease. Here, we combined single‐cell analysis, fate mapping, parabiosis, and computational modeling to comprehensively examine myeloid subsets in distinct parts of the eye during homeostasis. This approach allowed us to identify myeloid subsets displaying diverse transcriptional states. During choroidal neovascularization, a typical hallmark of neovascular age‐related macular degeneration (AMD), we recognized disease‐specific macrophage subpopulations with distinct molecular signatures. Our results highlight the heterogeneity of myeloid subsets and their dynamics in the eye that provide new insights into the innate immune system in this organ which may offer new therapeutic targets for ophthalmological diseases., Single‐cell RNA‐sequencing reveals heterogeneity of macrophages in the cornea, ciliary body and retina under homeostatic conditions.

Statins induce plaque regression characterized by reduced macrophage content in humans, but the underlying mechanisms remain speculative. Studying the translational APOE*3-Leiden.CETP mouse model with a humanized lipoprotein metabolism, we find that systemic cholesterol lowering by oral atorvastatin or dietary restriction inhibits monocyte infiltration, and reverses macrophage accumulation in atherosclerotic plaques. Contrary to current believes, none of (1) reduced monocyte influx (studied by cell fate mapping in thorax-shielded irradiation bone marrow chimeras), (2) enhanced macrophage egress (studied by fluorescent bead labeling and transfer), or (3) atorvastatin accumulation in murine or human plaque (assessed by mass spectrometry) could adequately account for the observed loss in macrophage content in plaques that undergo phenotypic regression. Instead, suppression of local proliferation of macrophages dominates phenotypic plaque regression in response to cholesterol lowering: the lower the levels of serum LDL-cholesterol and lipid contents in murine aortic and human carotid artery plaques, the lower the rates of in situ macrophage proliferation. Our study identifies macrophage proliferation as the predominant turnover determinant and an attractive target for inducing plaque regression. Supplementary Information The online version contains supplementary material available at 10.1007/s00395-020-00838-4.

Aims Mental stress substantially contributes to the initiation and progression of human disease, including cardiovascular conditions. We aim to investigate the underlying mechanisms of these contributions since they remain largely unclear. Methods and results Here, we show in humans and mice that leucocytes deplete rapidly from the blood after a single episode of acute mental stress. Using cell-tracking experiments in animal models of acute mental stress, we found that stress exposure leads to prompt uptake of inflammatory leucocytes from the blood to distinct tissues including heart, lung, skin, and, if present, atherosclerotic plaques. Mechanistically, we found that acute stress enhances leucocyte influx into mouse atherosclerotic plaques by modulating endothelial cells. Specifically, acute stress increases adhesion molecule expression and chemokine release through locally derived norepinephrine. Either chemical or surgical disruption of norepinephrine signalling diminished stress-induced leucocyte migration into mouse atherosclerotic plaques. Conclusion Our data show that acute mental stress rapidly amplifies inflammatory leucocyte expansion inside mouse atherosclerotic lesions and promotes plaque vulnerability., Graphical Abstract This study provides novel mechanistic insights into how acute mental stress fuels vascular inflammation and promotes plaque rupture. EC, endothelial cells; HPA, hypothalamic-pituitary-adrenal axis; MACS, macrophages; SAM, sympathetic-adrenal-medullary axis.

Background Bone morphogenic proteins (BMPs) are members of the TGFβ superfamily. They have distinct functions during hemostasis and play a central role in various pathologic conditions, including cardiovascular diseases. Recent findings demonstrate that BMPs are also expressed in platelets. However, their function is poorly understood. Here, we investigate the role of platelet BMP4 during vascular inflammation and vascular remodelling. Methods BMP4 floxed mice were crossed with PF4 Cre mice to generate platelet-specific deletion of BMP4 (BMP4Plt−/−). Intravital microscopy of mesenteric veins was performed to evaluate leukocyte adhesion upon stimulation with TNFα. Expression of adhesion molecules and chemokines were analysed by RT-PCR and Western Blot. P-selectin and platelet-leukocyte aggregates were evaluated using flow cytometry. For carotid wire injury, BMP4Plt−/− were further crossed with LDLr−/− mice (BMP4Plt−/−/LDLr−/−). At 8 weeks of age, BMP4Plt−/−/LDLr−/− mice and control littermates received a 2-week diet containing 15.8% wt/wt fat and 1.25% cholesterol. Carotid wire injury was performed at the age of 10 weeks. Re-endothelialisation and neointimal hyperplasia were evaluated. Results Platelet morphology and function did not differ between BMP4Plt−/− and control mice. Stimulation with TNFα resulted in increased rolling and adherence of leukocytes to the vessel wall which was reduced in BMP4Plt−/− mice (175±25 versus 50±7 rolling cells and 16±3 versus 7±2 adherent cells, respectively). Expression of P-selectin, adhesion molecules and the chemokines RANTES and PF-4 were reduced in BMP4Plt−/− mice. Platelet activation by thrombin was reduced in BMP4Plt−/− mice, resulting in diminished P-Selectin and JONA expression. Furthermore, monocyte infiltration and circulating leukocyte-platelet complexes were reduced in BMP4Plt−/− mice. Loss of platelet BMP4 prevented neointima formation after carotid wire injury (4.2x104±0.9x104μm2 versus 14.9x104±2.2x104μm2 in BMP4Plt−/−/LDLr−/− and control mice, respectively). Interestingly, endothelial regeneration after injury was decelerated in BMP4Plt−/− mice. This is further demonstrated in-vitro, where platelet BMP4 promoted endothelial cell proliferation and migration. Conclusion Platelet-BMP4 deficiency reduced vascular inflammation and ameliorated intima hyperplasia after wire injury. This is partly mediated by inhibition of platelet activation, reduced expression of adhesion molecules and inflammatory response. Our finding suggests that BMP4 is a promising target for the treatment of vascular inflammation and restenosis. Funding Acknowledgement Type of funding source: Private grant(s) and/or Sponsorship. Main funding source(s): Else-Kröner Fresenius Foundation; German Cardiac Society

Introduction Inflammation is essential for wound healing after myocardial infarction (MI). Leukocytes, especially neutrophils and monocytes, orchestrate removal of necrosis and regulation of tissue remodeling. Beside local recruitment from the blood, leukocyte supply via increased hematopoiesis is of major relevance for post-ischemic myocardial inflammation. Little is known about the pathways that carry the signals for increased demand for leukocytes from the site of injury to upstream hematopoietic stem cells (LSK) in the bone marrow (BM). In this study, we investigate the role of the P2Y12-ADP-receptor mediated regulation for emergency hematopoiesis after MI. Methods For standardized MI, a model of permanent coronary ligation was used in C57/Bl6 and P2Y12−/− mice. Changes in plasmatic ADP levels after MI were screened using ELISA, whereas the expression of the P2Y12-ADP-receptor in cell populations isolated from the BM was investigated by qPCR. CFU assays added further functional insight on hematopoietic proliferation in vitro. The effect of P2Y12 on the hematopoietic system after MI was investigated by inhibiting the P2Y12-receptor via prasugrel and compared to inhibition of the thromboxane pathway via acetylsalicylic acid (ASA). Proliferation of LSK in BM and leukocyte composition in blood, BM and infarct tissue after MI were assessed via FACS. Leukocyte composition in the infarcted myocardium was validated by immunohistochemistry. Finally, left ventricular function (LV-EF) and remodeling were investigated by echocardiography. Results 24 h after MI, we found a peak of plasmatic ADP levels. LSK as upstream hematopoietic progenitors in the BM express a P2Y12-receptor, which was validated on transcriptional and protein level. Whereas ADP stimulation of LSK led to significantly larger colony growth in vitro on the one hand, percentage of cycling LSK were significantly reduced 48 h after MI in P2Y12−/− mice compared to WT mice, assessed by Ki67/DAPI cell cycle analysis. Prasugrel treatment showed similar effects, translating into reduced numbers of downstream hematopoietic progenitors GMP and MDP 72 h after MI. Treatment with ASA however had no significant effect neither on cycling LSK nor progenitor populations. Consequently, decreased medullary hematopoiesis under P2Y12-inhibition led to reduced infiltration of inflammatory cells in the infarct tissue 7 days after MI, finally resulting in significantly improved outcome in terms of LV-EF 3 weeks after MI. Conclusion In this study, we demonstrate that P2Y12-mediated signaling is involved in emergency hematopoiesis after MI and advocates post-MI inflammation. In turn, inhibition of P2Y12-mediated signaling contributes to improved wound healing and prevention of adverse cardiac remodeling after MI, which adds a yet unknown mechanism to the success story of modern P2Y12-receptor blockers. Funding Acknowledgement Type of funding source: Public grant(s) – National budget only. Main funding source(s): Deutsche Forschungsgemeinschaft

Background Interferon regulatory factor (IRF) 5 is a transcription factor promoting inflammatory macrophage polarization (M1 type). Given the central role of macrophages in atherosclerotic plaque development we hypothesized that macrophage specific deletion of IRF5 will protect from atherosclerosis. Purpose Investigate whether intrinsic blockade of M1 macrophage polarization ameliorates atherosclerosis Methods Female ApoE−/−LysMCre/wtIRF5flox/floxand ApoE−/−LysMwt/wtIRF5flox/floxmice were fed a high cholesterol diet for 3 months, and atherosclerotic plaque size and compositions as well as inflammatory gene expression were analyzed. Mechanistically, IRF5-dependend bone marrow derived macrophage cytokine profiles were tested under M1 and M2 polarizing conditions. Aortic macrophage chimerism in irradiated ApoE−/− mice reconstituted with a mixture of CD45.1+ ApoE−/− (WT) and CD45.2+ ApoE−/− LysMCre/WtIRF5flox/flox(KO) bone marrow was evaluated to distinguish systemic from intra-plaque effects on monocyte/macrophage kinetics. Results Macrophage-specific IRF5 deficiency blunted LPS/IFNg-induced IL-1β and TNFα gene expression in vitro. In ApoE−/− mice, macrophage-specific IRF5 deficiency did not alter lesion size in the aortic root but significantly reduced macrophage and lipid contents by about 25% while increasing collagen deposition by over 30%. This was accompanied by relative reductions in gene expressions of pro-inflammatory (IL-1β, IL-6, IL-12) and increases in anti-inflammatory (Mertk, TGFβ, CD206) markers in atherosclerotic aortas of ApoE−/−LysMCre/wtIRF5flox/floxmice. When competing with IRF5 deficient cells in mixed irradiation bone marrow chimeras, IRF5 competent macrophages showed an advantage in accumulating in atherosclerotic aortas as disease progressed independent of monocyte recruitment. Conclusion Transcription factor IRF5 promotes a pro-inflammatory response in macrophages leading to vulnerable plaque formation and plaque destabilization, providing genetic evidence for targeting macrophage polarization in atherosclerosis. Funding Acknowledgement Type of funding source: Public grant(s) – National budget only. Main funding source(s): DFG

Background Atherosclerosis is accompanied by an imbalance between resolving and pro-inflammatory lipid mediators. Targeting lipid signaling pathways might offer a new anti-inflammatory therapy for improving the clinical outcome in cardiovascular disease patients. We considered lysophosphatidylinositol (LPI) and its receptor G protein-coupled receptor (GPR)55 as a potential modulator of atherosclerosis. Its role in regulating atherosclerosis and B cell function is unknown. Hypothesis We assessed the hypothesis that GPR55 signaling causally affects atherosclerosis and whether it has a specific role in regulating B cell function in this disease. Methods Atherosclerotic plaques were compared between apolipoprotein E deficient (ApoE−/−) and ApoE−/−Gpr55−/− mice after 4 to 16 weeks Western Diet (WD; 0.15% cholesterol; n=12–15 per group). To specifically test the role of B cell GPR55 in atherosclerosis, we generated mixed chimeras by lethally irradiating low density lipoprotein receptor deficient (Ldlr−/−) mice and reconstituting with a mixture of μMT and wildtype (control) or μMT and Gpr55−/− bone marrow cells. Circulating B cells were sorted and bulk RNA sequencing analysis was performed. We performed lipid and immunostainings of murine aortic root plaques, qPCR and ELISA of tissue lysates, as well as multiplex analysis of plasma immunoglobulins. Leukocyte plasma and tissue counts were determined by flow cytometry. Results GPR55 expression in mouse and human atherosclerotic plaques was detected by immunostaining. Furthermore, we confirmed murine Gpr55 mRNA expression on sorted circulating B220+B cells via qPCR, which was higher compared to CD3+ T cells, while CD11+ myeloid cells as well as NK cells had only low Gpr55 mRNA levels. ApoE−/−Gpr55−/− mice had significantly larger plaques after 4&16 weeks WD compared to ApoE−/− controls, with more pronounced body weight increases and higher cholesterol levels at the 16 weeks WD time point. In addition, global Gpr55 deficiency resulted in enhanced aortic pro-inflammatory cytokine mRNA expression (IL-1β, IL-6, TNFα) and a massively upregulated IgG1 plasma levels and increased percentages of splenic germinal center and plasma cells. B-cell RNA-seq analysis showed 460 differential expressed regulated genes in the ApoE−/−Gpr55−/− compared to ApoE−/−. The main pathways affected were calcium ion transport, immunoglobulin production, negative regulation of phosphorylation, and cellular component morphogenesis, suggesting a dsysregulation of B cell function. B cell specific Gpr55 deficiency blunted the metabolic effects on body weight and cholesterol, but still translated in larger atherosclerotic plaques and elevated plasma IgG levels compared to the respective controls. Conclusion Both global and B cell-restricted Gpr55 deficiency promotes atherosclerosis and is associated with a more pro-inflammatory phenotype. Our findings suggest a novel role for GPR55 in regulating B cell development and function. Funding Acknowledgement Type of funding source: Public grant(s) – National budget only. Main funding source(s): Deutsche Forschungsgemeinschaft (DFG)

Optogenetic approaches have evolved as potent means to investigate cardiac electrophysiology, with research ranging from the study of arrhythmia mechanisms to effects of cardiac innervation and heterocellular structural and functional interactions, both in healthy and diseased myocardium. Most commonly, these studies use channelrhodopsin-2 (ChR2)-expressing murine models that enable light-activated depolarization of the target cell population. However, each newly generated mouse line requires thorough characterization, as cell-type specific ChR2 expression cannot be taken for granted, and the electrophysiological response of its activation in the target cell should be evaluated. In this chapter, we describe detailed protocols for assessing ChR2 specificity using immunohistochemistry, isolation of specific cell populations to analyze electrophysiological effects of ChR2 activation with the patch-clamp technique, and whole-heart experiments to assess in situ effects of optical stimulation.