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Abstract Gravitational changes between micro- and hypergravity cause several adaptations and alterations in the human body. Besides muscular atrophy and immune system impairment, effects on the circulatory system have been described, which can be associated with a wide range of blood biomarker changes. This study examined nine individuals (seven males, two females) during a parabolic flight campaign (PFC). Thirty-one parabolas were performed in one flight day, resulting in ~22 s of microgravity during each parabola. Each participant was subjected to a single flight day with a total of 31 parabolas, totaling 11 min of microgravity during one parabolic flight. Before and after (1 hour (h) and 24 h), the flights blood was sampled to examine potential gravity-induced changes of circulating plasma proteins. Proximity Extension Assay (PEA) offers a proteomic solution, enabling the simultaneous analysis of a wide variety of plasma proteins. From 2925 unique proteins analyzed, 251 (8.58%) proteins demonstrated a differential regulation between baseline, 1 h and 24 h post flight. Pathway analysis indicated that parabolic flights led to altered levels of proteins associated with vesicle organization and apoptosis up to 24 h post microgravity exposure. Varying gravity conditions are associated with poorly understood physiological changes, including stress responses and fluid shifts. We provide a publicly available library of gravity-modulated circulating protein levels illustrating numerous changes in cellular pathways relevant for inter-organ function and communication.

Here, we provide a concise overview of recent developments in the identification of immunogenic epitopes in human apolipoprotein B-100 for immunization against atherosclerotic cardiovascular disease. Major steps forward toward a clinical application of vaccines include the design of humanized mouse models, tetramer-based identification of antigen-specific T cells, and novel analysis tools, such as single-cell RNA sequencing and cytometry by time of flight, to assess temporal and spatial changes in immune cells in atherosclerotic cardiovascular disease.

Background/Objectives: Acute myocardial infarction (AMI), characterized by irreversible heart muscle damage and impaired cardiac function caused by myocardial ischemia, is a leading cause of global mortality. The damage associated with reperfusion, particularly mitochondrial dysfunction and reactive oxygen species (ROS) formation, has emerged as a crucial factor in the pathogenesis of cardiac diseases, leading to the recognition of mitochondrial proteins as potential markers for myocardial damage. This study aimed to identify differentially expressed proteins based on the type of cardiac injury, in particular those with and without reperfusion. Methods: Male C57Bl/6J mice were either left untreated, sham-operated, received non-reperfused AMI, or reperfused AMI. Twenty-four hours after the procedures, left ventricular (LV) function and morphological changes including infarct size were determined using echocardiography and triphenyl tetrazolium chloride (TTC) staining, respectively. In addition, plasma was isolated and subjected to untargeted mass spectrometry and, further on, the ELISA-based validation of candidate proteins. Results: We identified mitochondrial creatine kinase 2 (Ckmt2) as a differentially regulated protein in plasma of mice with reperfused but not non-reperfused AMI. Elevated levels of Ckmt2 were significantly associated with infarct size and impaired LV function following reperfused AMI, suggesting a specific involvement in reperfusion damage. Conclusions: Our study highlights the potential of plasma Ckmt2 as a biomarker for assessing reperfusion injury and its impact on cardiac function and morphology in the acute phase of MI.

Human monocytes can be subdivided into phenotypically and functionally different classical, intermediate and non-classical monocytes according to the cell surface expression of CD14 and CD16. A precise identification and characterisation of monocyte subsets is necessary to unravel their role in inflammatory diseases. Here, we compared three different flow cytometric strategies (A–C) and found that strategy C, which included staining against CD11b, HLA-DR, CD14 and CD16, followed by several gating steps, most reliably identified monocyte subtypes in blood samples from healthy volunteers and from patients with stable coronary heart disease (CHD) or ST-elevation myocardial infarction (STEMI). Additionally, we established a fixation and permeabilisation protocol to enable the analysis of intracellular markers. We investigated the phagocytosis of lipid nanoparticles, the uptake of 2-NBD-glucose and the intracellular levels of CD74 and HLA-DM. This revealed that classical and intermediate monocytes from patients with STEMI showed the highest uptake of 2-NBD-glucose, whereas classical and intermediate monocytes from patients with CHD took up the largest amounts of lipid nanoparticles. Interestingly, intermediate monocytes had the highest expression level of HLA-DM. Taken together, we present a robust flow cytometric approach for the identification and functional characterisation of monocyte subtypes in healthy humans and patients with diseases.

Determination of infarct and scar size following myocardial infarction (MI) is commonly used to evaluate the efficacy of potential cardioprotective treatments in animal models. However, histological methods to determine morphological features in the infarcted heart have barely improved since implementation while still consuming large parts of the tissue and offering little options for parallel analyses. We aim to develop a new fluorescence technology for determining infarct area and area at risk that is comparable to 2,3,5-triphenyltetrazolium chloride (TTC) staining but allows for multiple analyses on the same heart tissue. For early and late time points following MI, we compared classical histochemical approaches with fluorescence staining methods. Reperfused MI was induced in male mice, the hearts were extracted 24 h, 7-, 21-, or 28-days later and fluorescently stained by combining Hoechst and phalloidin. This approach allowed for clear visualization of the infarct area, the area at ischemic risk and the remote area not affected by MI. The combined fluorescence staining correlated with the classic TTC/Evans Blue staining 24 h after MI (r = 0.8334). In later phases (>7 d) post-MI, wheat germ agglutinin (WGA) is equally accurate as classical Sirius Red (r = 0.9752), Masson’s (r = 0.9920) and Gomori’s Trichrome (r = 0.8082) staining for determination of scar size. Additionally, feasibility to co-localize fluorescence-stained immune cells in specific regions of the infarcted myocardium was demonstrated with this protocol. In conclusion, this new procedure for determination of post-MI infarct size is not inferior to classical TTC staining, yet provides substantial benefits, including the option for unbiased software-assisted analysis while sparing ample residual tissue for additional analyses. Overall, this enhances the data quality and reduces the required animal numbers consistent with the 3R concept of animal experimentation.

Abstract Background Smoking, alcohol abuse, and hypertension are – among others, potential risk factors for cardiovascular diseases. These risk factors generate oxidative stress and cause oxidative stress-induced DNA damage, resulting in cellular senescence and senescence-associated secretory phenotype (SASP). The SASP factors in feed-forward response exacerbate inflammation and cause tissue remodeling, resulting in atherosclerotic plaque formation and rupture. Results Colchicine inhibited ROS generation and mitigated oxidative stress-induced DNA damage. It dampened oxidative stress-induced endothelial cell senescence and improved the expression of DNA repair protein KU80 and aging marker Lamin B1. The drug attenuated the expression of senescence marker P21 at mRNA and protein levels. The pathway analysis showed that colchicine inhibited NF-κB and MAPKs pathways and subdued mTOR activation. Colchicine also attenuated mRNA expression of interleukin (IL)-1β, IL-6, IL-8, MCP-1, ICAM-1, and E-selectin. Furthermore, colchicine reduced the mRNA and protein expression of matrix metalloproteinase (MMP-2). Conclusion In summary, colchicine blocked oxidative stress-induced senescence and SASP by inhibiting the activation of NF-κB and MAPKs pathways. Graphical Abstract

Abstract 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.

BackgroundAn abdominal aortic aneurysm (AAA) is a life-threatening cardiovascular disease. Although its pathogenesis is still poorly understood, recent evidence suggests that AAA displays autoimmune disease characteristics. Particularly, T cells responding to AAA-related antigens in the aortic wall may contribute to an initial immune response. Single-cell RNA (scRNA) T cell receptor (TCR) and B cell receptor (BCR) sequencing is a powerful tool for investigating clonality. However, difficulties such as limited numbers of isolated cells must be considered during implementation and data analysis, making biological interpretation challenging. Here, we perform a representative single-cell immune repertoire analysis in experimental murine AAA and show a reliable bioinformatic processing pipeline highlighting opportunities and limitations of this approach.MethodsWe performed scRNA TCR and BCR sequencing of isolated lymphocytes from the infrarenal aorta of male C57BL/6J mice 3, 7, 14, and 28 days after AAA induction via elastase perfusion of the aorta. Sham-operated mice at days 3 and 28 and non-operated mice served as controls.ResultsComparison of complementarity-determining region (CDR3) length distribution of 179 B cells and 796 T cells revealed neither differences between AAA and control nor between the disease stages. We found no clonal expansion of B cells in AAA. For T cells, we identified several clones in 11 of 16 AAA samples and one of eight control samples. Immune receptor repertoire comparison indicated that only a few clones were shared between the individual AAA samples. The most frequently used V-genes in the TCR beta chain in AAA were TRBV3, TRBV19, and the splicing variant TRBV12-2 + TRBV13-2.ConclusionWe found no clonal expansion of B cells but evidence for clonal expansion of T cells in elastase-induced AAA in mice. Our findings imply that a more precise characterization of TCR and BCR distribution requires a more extensive number of lymphocytes to prevent undersampling and potentially detect rare clones. Thus, further experiments are necessary to confirm our findings. In summary, this paper examines TCR and BCR sequencing results, identifies limitations and pitfalls, and offers guidance for future studies.

Myasthenia gravis (MG) is a prototypical autoimmune disease of the neuromuscular junction (NMJ). The study of the underlying pathophysiology has provided novel insights into the interplay of autoantibodies and complement-mediated tissue damage. Experimental autoimmune myasthenia gravis (EAMG) emerged as a valuable animal model, designed to gain further insight and to test novel therapeutic approaches for MG. However, the availability of native acetylcholine receptor (AChR) protein is limited favouring the use of recombinant proteins. To provide a simplified platform for the study of MG, we established a model of EAMG using a recombinant protein containing the immunogenic sequence of AChR in mice. This model recapitulates key features of EAMG, including fatigable muscle weakness, the presence of anti-AChR-antibodies, and engagement of the NMJ by complement and a reduced NMJ density. Further characterization of this model demonstrated a prominent B cell immunopathology supported by T follicular helper cells. Taken together, the herein-presented EAMG model may be a valuable tool for the study of MG pathophysiology and the pre-clinical testing of therapeutic applications.

Abstract Background Peroxisome proliferator-activated receptor gamma coactivator-1 alpha (PGC-1α) acts as a transcriptional coactivator and regulates mitochondrial function. Various isoforms are generated by alternative splicing and differentially regulated promoters. In the heart, total PGC-1α deficiency knockout leads to dilatative cardiomyopathy, but knowledge on the complexity of cardiac isoform expression of PGC-1α remains sparse. Thus, this study aims to generate a reliable dataset on cardiac isoform expression pattern by long-read mRNA sequencing, followed by investigation of differential regulation of PGC-1α isoforms under metabolic and ischemic stress, using high-fat-high-sucrose-diet-induced obesity and a murine model of myocardial infarction. Results Murine (C57Bl/6J) or human heart tissue (obtained during LVAD-surgery) was used for long-read mRNA sequencing, resulting in full-length transcriptomes including 58,000 mRNA isoforms with 99% sequence accuracy. Automatic bioinformatic analysis as well as manual similarity search against exonic sequences leads to identification of putative coding PGC-1α isoforms, validated by PCR and Sanger sequencing. Thereby, 12 novel transcripts generated by hitherto unknown splicing events were detected. In addition, we postulate a novel promoter with homologous and strongly conserved sequence in human heart. High-fat diet as well as ischemia/reperfusion (I/R) injury transiently reduced cardiac expression of PGC-1α isoforms, with the most pronounced effect in the infarcted area. Recovery of PGC-1α-isoform expression was even more decelerated when I/R was performed in diet-induced obese mice. Conclusions We deciphered for the first time a complete full-length transcriptome of the murine and human heart, identifying novel putative PGC-1α coding transcripts including a novel promoter. These transcripts are differentially regulated in I/R and obesity suggesting transcriptional regulation and alternative splicing that may modulate PGC-1α function in the injured and metabolically challenged heart.

BackgroundIn acute myocardial infarction and heart failure, anemia is associated with adverse clinical outcomes. Endothelial dysfunction (ED) is characterized by attenuated nitric oxide (NO)-mediated relaxation responses which is poorly studied in chronic anemia (CA). We hypothesized that CA is associated with ED due to increased oxidative stress in the endothelium.MethodsCA was induced by repeated blood withdrawal in male C57BL/6J mice. Flow-Mediated Dilation (FMD) responses were assessed in CA mice using ultrasound-guided femoral transient ischemia model. Tissue organ bath was used to assess vascular responsiveness of aortic rings from CA mice, and in aortic rings incubated with red blood cells (RBCs) from anemic patients. In the aortic rings from anemic mice, the role of arginases was assessed using either an arginase inhibitor (Nor-NOHA) or genetic ablation of arginase 1 in the endothelium. Inflammatory changes in plasma of CA mice were examined by ELISA. Expression of endothelial NO synthase (eNOS), inducible NO synthase (iNOS), myeloperoxidase (MPO), 3-Nitrotyrosine levels, and 4-Hydroxynonenal (4-HNE) were assessed either by Western blotting or immunohistochemistry. The role of reactive oxygen species (ROS) in ED was assessed in the anemic mice either supplemented with N-Acetyl cysteine (NAC) or by in vitro pharmacological inhibition of MPO.ResultsThe FMD responses were diminished with a correlation to the duration of anemia. Aortic rings from CA mice showed reduced NO-dependent relaxation compared to non-anemic mice. RBCs from anemic patients attenuated NO-dependent relaxation responses in murine aortic rings compared to non-anemic controls. CA results in increased plasma VCAM-1, ICAM-1 levels, and an increased iNOS expression in aortic vascular smooth muscle cells. Arginases inhibition or arginase1 deletion did not improve ED in anemic mice. Increased expression of MPO and 4-HNE observed in endothelial cells of aortic sections from CA mice. NAC supplementation or inhibition of MPO improved relaxation responses in CA mice.ConclusionChronic anemia is associated with progressive endothelial dysfunction evidenced by activation of the endothelium mediated by systemic inflammation, increased iNOS activity, and ROS production in the arterial wall. ROS scavenger (NAC) supplementation or MPO inhibition are potential therapeutic options to reverse the devastating endothelial dysfunction in chronic anemia.

Abdominal aortic aneurysm (AAA) is a common disease and highly lethal if untreated. The progressive dilatation of the abdominal aorta is accompanied by degradation and remodeling of the vessel wall due to chronic inflammation. Pannexins represent anion-selective channels and play a crucial role in non-vesicular ATP release to amplify paracrine signaling in cells. Thus, pannexins are involved in many (patho-) physiological processes. Recently, Panx1 channels were identified to be significantly involved in abdominal aortic aneurysm formation through endothelial derived Panx1 regulated inflammation and aortic remodeling. In platelets, Panx1 becomes activated following activation of glycoprotein (GP) VI. Since platelets play a role in cardiovascular diseases including abdominal aortic aneurysm, we analyzed the contribution of platelet Panx1 in the progression of abdominal aortic aneurysm. We detected enhanced Panx1 plasma levels in abdominal aortic aneurysm patients. In experimental abdominal aortic aneurysm using the pancreatic porcine elastase (PPE) mouse model, a major contribution of platelet Panx1 channels in platelet activation, pro-coagulant activity of platelets and platelet-mediated inflammation has been detected. In detail, platelets are important for the migration of neutrophils into the aortic wall induced by direct cell interaction and by activation of endothelial cells. Decreased platelet activation and inflammation did not affect ECM remodeling or wall thickness in platelet-specific Panx1 knock-out mice following PPE surgery. Thus, aortic diameter expansion at different time points after elastase infusion of the aortic wall was unaltered in platelet-specific Panx1 deficient mice suggesting that the modulation of inflammation alone does not affect abdominal aortic aneurysm formation and progression. In conclusion, our data strongly supports the role of platelets in inflammatory responses in abdominal aortic aneurysm via Panx1 channels and adds important knowledge about the significance of platelets in abdominal aortic aneurysm pathology important for the establishment of an anti-platelet therapy for abdominal aortic aneurysm patients.

Inflammation is a key component in the pathogenesis of cardiovascular diseases causing a significant burden of morbidity and mortality worldwide. Recent research shows that mammalian target of rapamycin (mTOR) signaling plays an important role in the general and inflammation-driven mechanisms that underpin cardiovascular disease. mTOR kinase acts prominently in signaling pathways that govern essential cellular activities including growth, proliferation, motility, energy consumption, and survival. Since the development of drugs targeting mTOR, there is proven efficacy in terms of survival benefit in cancer and allograft rejection. This review presents current information and concepts of mTOR activity in myocardial infarction and atherosclerosis, two important instances of cardiovascular illness involving acute and chronic inflammation. In experimental models, inhibition of mTOR signaling reduces myocardial infarct size, enhances functional remodeling, and lowers the overall burden of atheroma. Aside from the well-known effects of mTOR inhibition, which are suppression of growth and general metabolic activity, mTOR also impacts on specific leukocyte subpopulations and inflammatory processes. Inflammatory cell abundance is decreased due to lower migratory capacity, decreased production of chemoattractants and cytokines, and attenuated proliferation. In contrast to the generally suppressed growth signals, anti-inflammatory cell types such as regulatory T cells and reparative macrophages are enriched and activated, promoting resolution of inflammation and tissue regeneration. Nonetheless, given its involvement in the control of major cellular pathways and the maintenance of a functional immune response, modification of this system necessitates a balanced and time-limited approach. Overall, this review will focus on the advancements, prospects, and limits of regulating mTOR signaling in cardiovascular disease.

Inflammaging is a potential risk factor for cardiovascular diseases. It results in the development of thrombosis and atherosclerosis. The accumulation of senescent cells in vessels causes vascular inflammaging and contributes to plaque formation and rupture. In addition to being an acquired risk factor for cardiovascular diseases, ethanol can induce inflammation and senescence, both of which have been implicated in cardiovascular diseases. In the current study, we used colchicine to abate the cellular damaging effects of ethanol on endothelial cells. Colchicine prevented senescence and averted oxidative stress in endothelial cells exposed to ethanol. It lowered the relative protein expression of aging and senescence marker P21 and restored expression of the DNA repair proteins KU70/KU80. Colchicine inhibited the activation of nuclear factor kappa B (NFκ-B) and mitogen activated protein kinases (MAPKs) in ethanol-treated endothelial cells. It reduced ethanol-induced senescence-associated secretory phenotype. In summary, we show that colchicine ameliorated the ethanol-caused molecular events, resulting in attenuated senescence and senescence-associated secretory phenotype in endothelial cells.