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Supplementary Figure Legend from Angiotensinogen Delays Angiogenesis and Tumor Growth of Hepatocarcinoma in Transgenic Mice

Angiotensinogen, a member of the serpin family, is involved in the suppression of tumor growth and metastasis. To investigate whether human angiotensinogen protects against tumor progression in vivo, we established an original bitransgenic model in which transgenic mice expressing human angiotensinogen (Hu-AGT-TG mice) were crossed with a transgenic mouse model of hepatocellular carcinoma (HCC-TG mice). Bitransgenic mice overexpressing human angiotensinogen (HCC/Hu-AGT-TG) had a significantly longer survival time than the HCC-TG mice and a reduction of both tumor growth and blood flow velocities in the liver. This antitumor effect of angiotensinogen is related to a reduced angiogenesis, impaired expression of endothelial arterial markers (active Notch4, Delta-like 4 ligand, and ephrin B2) with a decrease of arterial vessel density in HCC/Hu-AGT-TG mice liver. Overexpression of human angiotensinogen decreases angiogenesis, and prevents tumor sinusoids from remodeling and arterialization, thus delaying tumor progression in vivo. [Cancer Res 2009;69(7):2853–60]

Supplementary Figure 1 from Angiotensinogen Delays Angiogenesis and Tumor Growth of Hepatocarcinoma in Transgenic Mice

The autonomic sympathetic nervous system innervates peripheral resistance arteries, thereby controlling arterial diameter and modulating blood supply to organs and arterial tone. Despite its fundamental role in blood flow regulation and adaptive response of the cardiovascular system to challenging situations, how sympathetic arterial innervation develops remains poorly understood.We here show that sympathetic arterial innervation is regulated by the axonal guidance molecule EphrinA4 in arterial Smooth Muscle Cells (SMCs), which repels sympathetic axons via the EphA4 receptor. Specific inactivation of EphA4 in sympathetic axons induced a loss of repulsion and increased sympathetic innervation of peripheral arteries throughout life. Functional consequences were a significant increase in arterial tone (resistivity and vasoconstriction), leading to an elevated systemic arterial blood pressure that reached to hypertension under stressful circumstances. These findings identify a novel pathway that negatively regulates sympathetic arterial innervation, and could participate to the appearance of idiopathic resistant hypertension.

To explore differences in the clinical management of men and women in the 5 years after detecting a solitary pulmonary nodule (SPN) by chest radiograph or CT in routine clinical practice. We followed up 545 men and 347 women with an SPN detected by chest radiograph or CT in a retrospective cohort of 25,422 individuals undergoing routine thoracic imaging in 2010–2011. We compared the frequency of each management strategy (no further test, immediate intervention or follow up) according to sex by means of chi-squared. We estimated the relative risk of women versus men of having been followed up instead of an immediate intervention using multivariate logistic regression. We compared by sex the time between detection of the nodule and lung cancer diagnosis, the time between diagnosis and death by means of Mann-Whitney U test and the cumulative effective dose of radiation in each management strategy by means of t test. Women were more likely than men to have follow-up rather than immediate intervention (aRR = 1.8, CI 1.3–2.7, p = 0.002), particularly in those who underwent CT (aRR = 4.2, CI 1.9–9.3, p

AimsThe cardioprotective effects of human induced pluripotent stem cell-derived cardiovascular progenitor cells (CPC) are largely mediated by the paracrine release of extracellular vesicles (EV). We aimed to assess the immunological behaviour of EV-CPC, which is a prerequisite for their clinical translation.Methods and resultsFlow cytometry demonstrated that EV-CPC expressed very low levels of immune relevant molecules including HLA Class I, CD80, CD274 (PD-L1), and CD275 (ICOS-L); and moderate levels of ligands of the natural killer (NK) cell activating receptor, NKG2D. In mixed lymphocyte reactions, EV-CPC neither induced nor modulated adaptive allogeneic T cell immune responses. They also failed to induce NK cell degranulation, even at high concentrations. These in vitro effects were confirmed in vivo as repeated injections of EV-CPC did not stimulate production of immunoglobulins or affect the interferon (IFN)-γ responses from primed splenocytes. In a mouse model of chronic heart failure, intra-myocardial injections of EV-CPC, 3 weeks after myocardial infarction, decreased both the number of cardiac pro-inflammatory Ly6Chigh monocytes and circulating levels of pro-inflammatory cytokines (IL-1α, TNF-α, and IFN-γ). In a model of acute infarction, direct cardiac injection of EV-CPC 2 days after infarction reduced pro-inflammatory macrophages, Ly6Chigh monocytes, and neutrophils in heart tissue as compared to controls. EV-CPC also reduced levels of pro-inflammatory cytokines IL-1α, IL-2, and IL-6, and increased levels of the anti-inflammatory cytokine IL-10. These effects on human macrophages and monocytes were reproduced in vitro; EV-CPC reduced the number of pro-inflammatory monocytes and M1 macrophages, while increasing the number of anti-inflammatory M2 macrophages.ConclusionsEV-CPC do not trigger an immune response either in in vitro human allogeneic models or in immunocompetent animal models. The capacity for orienting the response of monocyte/macrophages towards resolution of inflammation strengthens the clinical attractiveness of EV-CPC as an acellular therapy for cardiac repair.

Background: Extracellular vesicles (EV) mediate the therapeutic effects of stem cells but it is unclear whether this involves cardiac regeneration mediated by endogenous cardiomyocyte proliferation. Methods: Bi-transgenic MerCreMer/ZEG (n = 15/group) and Mosaic Analysis With Double Markers (MADM; n = 6/group) mouse models underwent permanent coronary artery ligation and received, 3 weeks later, 10 billion EV (from human iPS-derived cardiovascular progenitor cells [CPC]), or saline, injected percutaneously under echo guidance in the peri-infarcted myocardium. Endogenous cardiomyocyte proliferation was tracked by EdU labeling and biphoton microscopy. Other end points, including cardiac function (echocardiography and MRI), histology and transcriptomics were blindly assessed 4-6 weeks after injections. Results: There was no proliferation of cardiomyocytes in either transgenic mouse strains. Nevertheless, EV improved cardiac function in both models. In MerCreMer/ZEG mice, LVEF increased by 18.3 ± 0.2% between baseline and the end-study time point in EV-treated hearts which contrasted with a decrease by 2.3 ± 0.2% in the PBS group; MADM mice featured a similar pattern as intra-myocardial administration of EV improved LVEF by 13.3 ± 0.16% from baseline whereas it decreased by 14.4 ± 0.16% in the control PBS-injected group. This functional improvement was confirmed by MRI and associated with a reduction in infarct size, the decreased expression of several pro-fibrotic genes and an overexpression of the anti-fibrotic miRNA 133-a1 compared to controls. Experiments with an anti-miR133-a demonstrated that the cardio-reparative effects of EV were partly abrogated. Conclusions: EV-CPC do not trigger cardiomyocyte proliferation but still improve cardiac function by other mechanisms which may include the regulation of fibrosis.

Background: Ischemic cardiovascular diseases, particularly acute myocardial infarction (MI), is one of the leading causes of mortality worldwide. Indoleamine 2, 3-dioxygenase 1 (IDO) catalyzes 1 rate-limiting step of L-tryptophan metabolism, and emerges as an important regulator of many pathological conditions. We hypothesized that IDO could play a key role to locally regulate cardiac homeostasis after MI. Methods: Cardiac repair was analyzed in mice harboring specific endothelial or smooth muscle cells or cardiomyocyte or myeloid cell deficiency of IDO and challenged with acute myocardial infarction. Results: We show that kynurenine generation through IDO is markedly induced after MI in mice. Total genetic deletion or pharmacological inhibition of IDO limits cardiac injury and cardiac dysfunction after MI. Distinct loss of function of IDO in smooth muscle cells, inflammatory cells, or cardiomyocytes does not affect cardiac function and remodeling in infarcted mice. In sharp contrast, mice harboring endothelial cell–specific deletion of IDO show an improvement of cardiac function as well as cardiomyocyte contractility and reduction in adverse ventricular remodeling. In vivo kynurenine supplementation in IDO-deficient mice abrogates the protective effects of IDO deletion. Kynurenine precipitates cardiomyocyte apoptosis through reactive oxygen species production in an aryl hydrocarbon receptor–dependent mechanism. Conclusions: These data suggest that IDO could constitute a new therapeutic target during acute MI.

International audience; The triggering receptor expressed on myeloid cells 1 (TREM-1) drives inflammatory responses in several cardiovascular diseases but its role in abdominal aortic aneurysm (AAA) remains unknown. Our objective was to explore the role of TREM-1 in a mouse model of angiotensin II-induced (AngII-induced) AAA. TREM-1 expression was detected in mouse aortic aneurysm and colocalized with macrophages. Trem1 gene deletion (Apoe-/-Trem1-/-), as well as TREM-1 pharmacological blockade with LR-12 peptide, limited both AAA development and severity. Trem1 gene deletion attenuated the inflammatory response in the aorta, with a reduction of Il1b, Tnfa, Mmp2, and Mmp9 mRNA expression, and led to a decreased macrophage content due to a reduction of Ly6Chi classical monocyte trafficking. Conversely, antibody-mediated TREM-1 stimulation exacerbated Ly6Chi monocyte aorta infiltration after AngII infusion through CD62L upregulation and promoted proinflammatory signature in the aorta, resulting in worsening AAA severity. AngII infusion stimulated TREM-1 expression and activation on Ly6Chi monocytes through AngII receptor type I (AT1R). In human AAA, TREM-1 was detected and TREM1 mRNA expression correlated with SELL mRNA expression. Finally, circulating levels of sTREM-1 were increased in patients with AAA when compared with patients without AAA. In conclusion, TREM-1 is involved in AAA pathophysiology and may represent a promising therapeutic target in humans.

Introduction: Extracellular Vesicles (EV) recapitulate the benefits of cell therapy for heart repair. Their mechanism of action remains unsettled. Hypothesis: EV may contribute to heart repair by de novo cardiogenesis. Methods: To answer this question, we used 2 bi-transgenic mouse models: the fate-mapping MerCreMer/ZEG and the Mosaic Analysis With Double Markers (MADM). Myocardial infarction was induced by permanent coronary artery ligation. Those with a LVEF ≤ 45% were treated 3 weeks later with EV (from human iPS-derived cardiovascular progenitor cells; 10x10 9 particles) or PBS, injected under echo guidance in the peri-infarcted area (MerCreMer/ZEG: n=15/group and MADM: n=6/group). To track endogenous cardiomyocyte (CM) proliferation, we used EdU labeling in MerCreMer/ZEG delivered by osmotic pumps implanted for 7-10 days post-injection and biphoton microscopy in MADM models. Cardiac function was assessed 4-6 weeks after injection by echocardiography and MRI, blinded to treatment group. Hearts were then subjected to histological and transcriptomic analyses (qPCR and genome-wide microarray). Results: In PBS controls, EF remained stable over time in MerCreMer/ZEG mice and decreased from 34.5% ± 6.0% to 30.7% ± 7.5% in MADM mice by the end of the study. Conversely, EV injections increased EF from 32.1% ± 9.5% to 36.1% ± 7.45 % in MerCreMer/ZEG and from 36.2 %± 8.7% to 40.5% ± 8.9% in MADM mice. A significant difference in the change from baseline was found between EV and controls: 20.7% ± 10.5 % (p=0.048) and 28.0% ± 11.0 %, (p=0.045) for MerCreMer/ZEG and MADM groups, respectively. This improvement was confirmed by MRI in MerCreMer/ZEG mice (p=0.05). Improvement in EF was unrelated to the appearance of new CM, as shown by the absence of difference in TnT+/EdU+/GFP+ cell numbers and the lack of activation of the YAP/TAZ pathway between control and EV groups. However, EV reduced infarct size by 11.9% ± 5.75% (p=0.04), which was accompanied by decreased expression of 4 pro-fibrotic genes (Col1a2, Col3a1, Lox, Col1a2 by qPCR) in heart tissue and a 2.13X overexpression of the anti-fibrotic miRNA 133a-1 compared to controls (n=3/group; p=0.001). Conclusions: EV likely improve cardiac function by modulation of fibrosis rather than by de novo cardiogenesis.

Rationale: After myocardial infarction, neutrophils rapidly and massively infiltrate the heart, where they promote both tissue healing and damage. Objective: To characterize the dynamics of circulating and cardiac neutrophil diversity after infarction. Methods and results: We employed single-cell transcriptomics combined with cell surface epitope detection by sequencing to investigate temporal neutrophil diversity in the blood and heart after murine myocardial infarction. At day 1, 3, and 5 after infarction, cardiac Ly6G + (lymphocyte antigen 6G) neutrophils could be delineated into 6 distinct clusters with specific time-dependent patterning and proportions. At day 1, neutrophils were characterized by a gene expression profile proximal to bone marrow neutrophils ( Cd177 , Lcn2 , Fpr1 ), and putative activity of transcriptional regulators involved in hypoxic response ( Hif1a ) and emergency granulopoiesis ( Cebpb ). At 3 and 5 days, 2 major subsets of Siglecf hi (enriched for eg, Icam1 and Tnf ) and Siglecf low ( Slpi, Ifitm1 ) neutrophils were found. Cellular indexing of transcriptomes and epitopes by sequencing (CITE-seq) analysis in blood and heart revealed that while circulating neutrophils undergo a process of aging characterized by loss of surface CD62L and upregulation of Cxcr4 , heart infiltrating neutrophils acquired a unique SiglecF hi signature. SiglecF hi neutrophils were absent from the bone marrow and spleen, indicating local acquisition of the SiglecF hi signature. Reducing the influx of blood neutrophils by anti-Ly6G treatment increased proportions of cardiac SiglecF hi neutrophils, suggesting accumulation of locally aged neutrophils. Computational analysis of ligand/receptor interactions revealed putative pathways mediating neutrophil to macrophage communication in the myocardium. Finally, SiglecF hi neutrophils were also found in atherosclerotic vessels, revealing that they arise across distinct contexts of cardiovascular inflammation. Conclusions: Altogether, our data provide a time-resolved census of neutrophil diversity and gene expression dynamics in the mouse blood and ischemic heart at the single-cell level, and reveal a process of local tissue specification of neutrophils in the ischemic heart characterized by the acquisition of a SiglecF hi signature.

Acute myocardial infarction is a common condition responsible for heart failure and sudden death. Here, we show that following acute myocardial infarction in mice, CD8+ T lymphocytes are recruited and activated in the ischemic heart tissue and release Granzyme B, leading to cardiomyocyte apoptosis, adverse ventricular remodeling and deterioration of myocardial function. Depletion of CD8+ T lymphocytes decreases apoptosis within the ischemic myocardium, hampers inflammatory response, limits myocardial injury and improves heart function. These effects are recapitulated in mice with Granzyme B-deficient CD8+ T cells. The protective effect of CD8 depletion on heart function is confirmed by using a model of ischemia/reperfusion in pigs. Finally, we reveal that elevated circulating levels of GRANZYME B in patients with acute myocardial infarction predict increased risk of death at 1-year follow-up. Our work unravels a deleterious role of CD8+ T lymphocytes following acute ischemia, and suggests potential therapeutic strategies targeting pathogenic CD8+ T lymphocytes in the setting of acute myocardial infarction., Immune cells contribute to adverse remodeling following myocardial infarction. Here the authors show in mice and pigs that CD8+ lymphocytes release Granzyme B in the infarcted heart leading to cardiomyocyte death, enhanced inflammation and deterioration of cardiac function.

International audience; Ischemic cardiovascular diseases, particularly acute myocardial infarction (MI) is one of the leading cause of mortality worldwide. Indoleamine 2, 3-dioxygenase 1 (IDO) catalyzes one rate-limiting step of L-Tryptophan (Trp) metabolism, and emerges as an important regulator of many pathological conditions. We hypothesized that IDO could play a key role to locally regulate cardiac homeostasis after MI. METHODS: Cardiac repair was analyzed in mice harboring specific endothelial or smooth muscle cells or cardiomyocyte or myeloid cell deficiency of IDO and challenged with acute myocardial infarction. RESULTS: We show that Kynurenine (Kyn) generation through IDO is markedly induced after MI in mice. Total genetic deletion or pharmacological inhibition of IDO limits cardiac injury and cardiac dysfunction after MI. Distinct loss of function of IDO in smooth muscle cells, inflammatory cells, or cardiomyocytes does not impact cardiac function and remodeling in infarcted mice. In sharp contrast, mice harboring endothelial cell-specific deletion of IDO show an improvement of cardiac function, as well as cardiomyocyte contractility and reduction in adverse ventricular remodeling. In vivo Kyn supplementation in IDO-deficient mice abrogates the protective effects of IDO deletion. Notably, Kyn precipitates cardiomyocyte apoptosis through reactive oxygen species production in an aryl hydrocarbon receptor-dependent mechanism. CONCLUSIONS: These data suggest that IDO could constitute a new therapeutic target during acute MI.

Objective— To determine the consequences of specific inhibition of EGFR (epidermal growth factor receptor) in myeloid cells in atherosclerosis development. Approach and Results— Atherosclerotic lesion size was significantly reduced in irradiated Ldlr −/− mice reconstituted with LysM Cre+ Egfr lox/lox bone marrow, compared with chimeric Ldlr −/− mice reconstituted with LysM Cre− Egfr lox/lox bone marrow, after 4 (−43%; P P P Egfr in myeloid cells reduced TNF-α (tumor necrosis factor-α) and IL (interleukin)-6 production by stimulated macrophages but had no effect on IL-10 and IL-12p70 secretion. Finally, we found that myeloid deletion of Egfr limited cytoskeletal rearrangements and also lipid uptake by macrophages through a downregulation of the scavenger receptor CD36 (cluster of differentiation 36). Conclusions— Gene deletion of Egfr in myeloid cells limits IL-6 and TNF-α production, lipid uptake, and consecutively reduces atherosclerosis development.

This study aims to provide and analyze Francisco Martinez de la Rosa’s unpublished correspondence during his term as President of the Spanish Government in the 1834-1835 biennium, maintained with Amat di San Filippo Luigi, nuncio in Spain, and kept in the Vatican Secret Archives. Such correspondence consists of three letters with official mail format, although they could be considered rather confidential for their content. In these documents, the Spanish government strives, albeit unsuccessfully, to achieve recognition by Rome of Elizabeth II of Spain and his liberal regime. And last but not least, in this paper, it is also noteworthy to highlight the urgent need to fill the many vacant Dioceses in Spain.

The aim of this study was to review recent literature in order to provide updated values of the typical effective doses associated with the top 20 imaging tests for adults and children and for the most widely used set of weights (ICRP60) as well as for the most recent one (ICRP103). We performed a systematic research on radiation dosimetry in radiology published from 2007 onwards through the Medline, Embase and Cochrane Library Plus databases. We also included studies backed by scientific or governmental organizations. Other variables included: year and type of study (survey or descriptive), country, method and sample used for the measurement. Mean effective dose, minimum, maximum and standard deviation were calculated. We compared our results with previous evidence and with data from DDM2. We included 27 articles and 5 web references in the study. A total of 378 values from the 20 procedures included were obtained, 280 (74%) using ICRP60 and 98 (26%) using ICRP103. Effective doses for CT procedures in children were very similar to those for adults, with the exception of CT Trunk, but fluoroscopy procedures had consistently lower dose. There were differences between the current data with either ICRP60 or ICRP103, and the previous published data. In conclusion, we provided the best available evidence from literature to evaluate the effective dose received by each patient for the most typical examinations. According to the recommendations from the Report 154 and from the European Council Directive, these results could also be useful to estimate the range of average exposures to the population.

Introduction Myocardial infarction (MI) is a severe ischemic disease precipitating long-term adverse remodeling and heart failure. The chemokine CXCL12/SDF-1 is essential for cardiovascular system development and plays a prominent role in physio-pathological processes such as inflammation, angiogenesis and tissue fibrosis. In addition to the binding to its cognate receptors CXCR4 and CXCR7, CXCL12 interacts with heparan-sulfates (HS) which coordinate its biological activity. We have previously highlighted the essential role of CXCL12/HS interactions in vascular growth and remodeling in the setting of critical limb ischemia. In addition, studies in experimental model of MI revealed a protective role for the CXCL12α isoform, through the regulation of cardiomyocyte survival and recruitment of inflammatory cells. However, in mice, three CXCL12 isoforms (α, β and γ) have been identified and, among them, the CXCL12γ isoform shows an unchallenged ability to cooperate with HS, suggesting a putative pivotal role in tissue repair. Objectives The aim of the study is to analyze the role of CXCL12γ isoform and the importance of CXCL12/HS interactions in post-ischemic cardiac remodeling in an acute model of MI. Methods MI was induced by permanent ligation of the left ascending coronary artery in mice carrying a Cxcl12 gene mutation that precludes interactions with HS (Cxcl12Gagtm) and in Cxcl12γ knock-in animals (Cxcl12γ-KI) harboring CXCL12γ deficiency. Alternatively, the impact of CXCL12γ overexpression and the importance of its interactions with HS was also evaluated in wild-type (WT) mice receiving transcutaneous echo-guided injections of adenovirus encoding WT Cxcl12γ or HS-binding-disabled Cxcl12γ in cardiac tissue. Cardiac function and remodeling have been assessed through echocardiography analysis, evaluation of infarct size, interstitial fibrosis, vascular growth (capillary and arteriole densities) and inflammatory cell infiltration into the cardiac tissue. Results After MI, Cxcl12Gagtm and Cxcl12γ-KI animals exhibit reduction in cardiac function and adverse left ventricular remodeling when compared to their respective WT littermates. Interestingly, overexpression of CXCL12γ in WT mice cardiac restored cardiac function by reducing the size of the infarcted area, interstitial fibrosis and promoting vascular growth. In sharp contrast, HS–binding disabled CXCL12gamma mutants failed to improve cardiac function and to abrogate adverse left ventricular remodeling. Conclusion We show that CXCL12γ isoform plays an important role in the regulation of post-ischemic cardiac function and remodeling and that its interactions with HS are essential for adequate cardiac repair in the setting of acute MI.

Background Myocardial infarction (MI) is a severe ischemic disease responsible for heart failure and sudden death. Mature B lymphocytes have been shown to exacerbate tissue injury and deterioration of cardiac function after MI. However, the cellular and molecular mechanisms governing B cell deleterious effects in the ischemic milieu remain to be defined. Purpose In this study, we speculate that endogenous activation of the miR21/HIFα-related pathways mediates the effect of B lymphocytes on post-ischemic cardiac remodeling. Methods Acute MI was induced by permanent ligation of the left anterior descending artery in mice. Cardiac function and remodeling was determined by echocardiography and immunohistochemistry. Inflammatory cell number and phenotype were defined by FACS analysis. To evaluate the role of HIFα isoforms in B cells, we generated mice with B cell lineage specific (Cd79aCre/+) conditional deletion of HIF1α (HIF1αflox/flox), HIF2α (HIF2αflox/flox), or both isoforms (HIF1α-HIF2αflox/flox). Results Acute MI increased miR21 levels in B cells. miR21 deficient mice showed reduced B cell numbers in the spleen, blood and subsequently in the injured cardiac tissue. Transplantation of bone marrow derived cells isolated from miR21-deficient mice (miR21−/−) improved cardiac function and remodeling when compared to administration of wild-type (WT) bone marrow cells. Similarly, in Rag1−/− immunodeficient mice with acute MI, re-supplementation with miR21−/− B lymphocytes restored cardiac repair and function when compared to injection of WT B cells. miR21 abrogated PTEN contents and subsequently enhanced HIF1α levels in cultured B cells. B cell deletion of HIF1α, but not that of HIF2α, reduced B cell accumulation and improved cardiac function after MI. Mice, which were equally deficient in HIF1α and HIF2α, also exhibited abrogation of adverse ventricular remodeling and showed recovery of cardiac function after MI. Toll like receptor agonist, CpG, fostered the release of the monocyte chemo-attractant protein, Ccl7, in cultured WT B cells but not in miR21- or HIF1α- deficient B cells. Ccl7 circulating levels were also reduced in miR21−/− and Cd79aCre/+/HIF1α flox/flox animals after acute MI. Ccl7 down-regulation hampered Ly6Chigh monocyte infiltration in the ischemic myocardium, leading to decreased infarct size and interstitial fibrosis, supporting cardiac repair. Conclusion This work reveals a novel function for miR21/HIF1α related pathways in B lymphocyte dependent effect on cardiac function and remodeling in the setting of acute MI.

ObjectiveAfter myocardial infarction, neutrophils rapidly and massively infiltrate the heart, where they can promote both tissue healing and damage. Here, we investigated the dynamics of cardiac neutrophil heterogeneity after infarction.Methods and resultsWe employed single-cell transcriptomics (scRNA-seq) to investigate temporal neutrophil heterogeneity in the heart after murine myocardial infarction. At day 1, 3, and 5 after infarction, neutrophils could be delineated into six distinct clusters with specific time-dependent patterning and proportions. While the majority of neutrophils at day 1 were characterized by high expression of chemokines (e.g.Cxcl3,Ccl6), and putative activity of transcriptional regulators involved in hypoxic response (Hif1a) and emergency granulopoiesis (Cebpb), two major subsets ofSiglecfhi(enriched for e.g.Icam1andTnf) andSiglecflow(Slpi, Ifitm1) neutrophils were found at 3 and 5 days. Flow cytometry analysis confirmed the presence of LY6G+SIGLECFhiand LY6G+SIGLECFlowneutrophils in the heart from 3 days after infarction onwards. LY6G+SIGLECFhineutrophils were absent from the bone marrow, blood and spleen, suggesting local acquisition of surface SIGLECF. Acquisition of the SIGLECFhistate was paralleled by features of neutrophil ageing and activation (ICAM1hiCXCR4hiCD49dhiCD62Llow). scRNA-seq of atherosclerotic aortas revealed two neutrophil subsets with gene expression patterns reminiscent of the majorSiglecfhiandSiglecflowcardiac neutrophil subpopulations, revealing that these populations may be present across distinct contexts of cardiovascular inflammation.ConclusionAltogether, our data provide a time-resolved census of neutrophil diversity and gene expression dynamics in the mouse ischemic heart at the single-cell level, and suggests that temporal neutrophil heterogeneity is in part driven by local transition to a SIGLECFhistate.

The ECHO model was developed to expand access to medical care for populations with HCV infection in underserved areas. We aimed to compare HCV treatment outcomes in community‐based clinics with the Austral University Hospital (AUH) and to assess improvement in physician knowledge and skills. In October 2015, we established an HCV ECHO clinic at the AUH in Buenos Aires. To evaluate the impact of this programme, we conducted a prospective cohort study comparing treatment for HCV infection at the AUH with healthcare providers from different Argentinean provinces. A survey evaluating skills and competence in HCV care was administered, and results were compared. The primary endpoint was sustained virologic response (SVR) and under direct‐acting antivirals. Since the implementation of ECHO clinics, a total of 25 physicians participated in at least one session (median 10.0; IQR 3.0‐18.0). SVR rates (n = 437 patients) were 94.2% (95% CI 90.4‐96.8) in patients treated at AUH clinic (n = 227/242) and 96.4% (95% CI 92.7‐98.5) in those treated at ECHO sites (n = 188/195), with a nonsignificant difference between sites, 2.2% SVR difference (95% CI −0.24‐0.06; P = 0.4). We also found a significant improvement in all the evaluated skills and abilities. Replicating the ECHO model helped to improve participants´ skills in the management of HCV achieving similar SVR rates. ECHO model was demonstrated to be an effective intervention able to multiply and expand HCV treatment, a critical barrier to access to care that needs to be solved if we are committed with WHO goals to eliminate HCV by 2030 Fil: Mendizabal, Manuel. Gobierno de la Provincia de Buenos Aires. Hospital Universitario Austral; Argentina Fil: Ridruejo, Ezequiel. Gobierno de la Provincia de Buenos Aires. Hospital Universitario Austral; Argentina Fil: Ceballos, Susana. Gobierno de la Provincia de Jujuy. Hospital San Roque; Argentina Fil: Sixto, Marcela. Gobierno de la Provincia de Santa Fe. Hospital José M. Cullen; Argentina Fil: Billordo, Ariel. Gobierno de la Provincia del Chaco. Hospital Julio C. Perrando; Argentina Fil: Gadea, Claudia. Sanatorio Pasquini de la Provincia de Tucumán; Argentina Fil: Mengarelli, Silvia. Gobierno de la Provincia de Cordoba. Nuevo Hospital San Roque.; Argentina Fil: Alonso, Cristina. Gobierno de la Provincia de Buenos Aires. Hospital Universitario Austral; Argentina Fil: Palazzo, Ana. Gobierno de la Provincia de Tucumán. Hospital Padilla; Argentina Fil: De María, Luis. Gobierno de la Provincia de Santa Fe. Hospital José M. Cullen; Argentina Fil: Bruno, Andrés. Gobierno de la Provincia de Buenos Aires. Hospital Argerich; Argentina Fil: Perez, Daniela. Gobierno de la Provincia de Tucumán. Hospital Padilla; Argentina. Gobierno de la Provincia de Buenos Aires. Hospital Universitario Austral; Argentina Fil: Piñero, Federico. Gobierno de la Provincia de Buenos Aires. Hospital Universitario Austral; Argentina Fil: Deltrozzo, Verónica. Dual Medica de la Provincia de Santiago del Estero; Argentina Fil: Mendoza, Carlos. Gobierno de la Provincia de Río Cuarto. Hospital Central de Río Cuarto; Argentina Fil: Figueroa, Sebastián. Hospital Dr. Arturo Oñativia - Salta Capital.; Argentina Fil: Manero, Estela. Gobierno de la Provincia de Jujuy. Hospital Pablo Soria; Argentina Fil: Villa, Marina. Instituto Oulton de Córdoba; Argentina Fil: Barreyro, Fernando Javier. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Nordeste; Argentina Fil: Moreno, Valeria. Gobierno de la Provincia de Cordoba. Nuevo Hospital San Roque.; Argentina Fil: Vilar, José. Asociación Argentina para el Estudio de las Enfermedades del Hígado; Argentina Fil: Murga, Dolores. Gobierno de la Provincia de Tucumán. Hospital Padilla; Argentina Fil: Fernandez, Marcelo. Gobierno de la Provincia de Entre Ríos. Hospital Bicentenario; Argentina Fil: O´Flaherty, Martin. University of Liverpool; Reino Unido Fil: Arora, Sanjeev. University of New Mexico; Estados Unidos Fil: Silva, Marcelo. Hospital Universitario Austral; Argentina

Background: Defective systemic and local iron metabolism correlates with cardiac disorders. Hepcidin, a master iron sensor, actively tunes iron trafficking. We hypothesized that hepcidin could play a key role to locally regulate cardiac homeostasis after acute myocardial infarction. Methods: Cardiac repair was analyzed in mice harboring specific cardiomyocyte or myeloid cell deficiency of hepcidin and challenged with acute myocardial infarction. Results: We found that the expression of hepcidin was elevated after acute myocardial infarction and the specific deletion of hepcidin in cardiomyocytes failed to improve cardiac repair and function. However, transplantation of bone marrow–derived cells from hepcidin-deficient mice ( Hamp −/− ) or from mice with specific deletion of hepcidin in myeloid cells (LysM CRE/+ / Hamp f/f ) improved cardiac function. This effect was associated with a robust reduction in the infarct size and tissue fibrosis in addition to favoring cardiomyocyte renewal. Macrophages lacking hepcidin promoted cardiomyocyte proliferation in a prototypic model of apical resection–induced cardiac regeneration in neonatal mice. Interleukin (IL)–6 increased hepcidin levels in inflammatory macrophages. Hepcidin deficiency enhanced the number of CD45 + /CD11b + /F4/80 + /CD64 + /MHCII Low /chemokine (C–C motif) receptor 2 (CCR2) + inflammatory macrophages and fostered signal transducer and activator of transcription factor-3 (STAT3) phosphorylation, an instrumental step in the release of IL-4 and IL-13. The combined genetic suppression of hepcidin and IL-4/IL-13 in macrophages failed to improve cardiac function in both adult and neonatal injured hearts. Conclusions: Hepcidin refrains macrophage-induced cardiac repair and regeneration through modulation of IL-4/IL-13 pathways.

Ngkelo et al. use a mast cell–deficient mouse model to reveal a protective role of mast cells in myocardial infarction, through regulation of the cardiac contractile machinery., Acute myocardial infarction (MI) is a severe ischemic disease responsible for heart failure and sudden death. Inflammatory cells orchestrate postischemic cardiac remodeling after MI. Studies using mice with defective mast/stem cell growth factor receptor c-Kit have suggested key roles for mast cells (MCs) in postischemic cardiac remodeling. Because c-Kit mutations affect multiple cell types of both immune and nonimmune origin, we addressed the impact of MCs on cardiac function after MI, using the c-Kit–independent MC-deficient (Cpa3Cre/+) mice. In response to MI, MC progenitors originated primarily from white adipose tissue, infiltrated the heart, and differentiated into mature MCs. MC deficiency led to reduced postischemic cardiac function and depressed cardiomyocyte contractility caused by myofilament Ca2+ desensitization. This effect correlated with increased protein kinase A (PKA) activity and hyperphosphorylation of its targets, troponin I and myosin-binding protein C. MC-specific tryptase was identified to regulate PKA activity in cardiomyocytes via protease-activated receptor 2 proteolysis. This work reveals a novel function for cardiac MCs modulating cardiomyocyte contractility via alteration of PKA-regulated force–Ca2+ interactions in response to MI. Identification of this MC-cardiomyocyte cross-talk provides new insights on the cellular and molecular mechanisms regulating the cardiac contractile machinery and a novel platform for therapeutically addressable regulators.

Supplemental Digital Content is available in the text., Background— In infarcted heart, improper clearance of dying cells by activated neighboring phagocytes may precipitate the transition to heart failure. We analyzed the coordinated role of 2 major mediators of efferocytosis, the myeloid-epithelial-reproductive protein tyrosine kinase (Mertk) and the milk fat globule epidermal growth factor (Mfge8), in directing cardiac remodeling by skewing the inflammatory response after myocardial infarction. Methods and Results— We generated double-deficient mice for Mertk and Mfge8 (Mertk−/−/Mfge8−/−) and challenged them with acute coronary ligature. Compared with wild-type, Mertk-deficient (Mertk−/−), or Mfge8-deficient (Mfge8−/−) animals, Mertk−/−/Mfge8−/− mice displayed greater alteration in cardiac function and remodeling. Mertk and Mfge8 were expressed mainly by cardiac Ly6CHigh and Low monocytes and macrophages. In parallel, Mertk−/−/Mfge8−/− bone marrow chimeras manifested increased accumulation of apoptotic cells, enhanced fibrotic area, and larger infarct size, as well as reduced angiogenesis. We found that the abrogation of efferocytosis affected neither the ability of circulating monocytes to infiltrate cardiac tissue nor the number of resident Ly6CHigh and Ly6CHow monocytes/macrophages populating the infarcted milieu. In contrast, combined Mertk and Mfge8 deficiency in Ly6CHigh/Ly6CLow monocytes/macrophages either obtained from in vitro differentiation of bone marrow cells or isolated from infarcted hearts altered their capacity of efferocytosis and subsequently blunted vascular endothelial growth factor A (VEGFA) release. Using LysMCre+/VEGFAfl/fl mice, we further identified an important role for myeloid-derived VEGFA in improving cardiac function and angiogenesis. Conclusions— After myocardial infarction, Mertk- and Mfge8-expressing monocyte/macrophages synergistically engage the clearance of injured cardiomyocytes, favoring the secretion of VEGFA to locally repair the dysfunctional heart.

Background & Aim Introduction Cell therapy for chronic heart failure has demonstrated that Cardiac Progenitor Cells (CPCs) delivered to the heart exert their beneficial effects through a paracrine mechanism harnessing endogenous repair pathways largely mediated by extracellular vesicles (EVs). We used a bi-transgenic fate-mapping MerCreMer/ZEG mouse model to assess whether the cardioprotection could be partly explained by de novo cardiogenesis. Methods, Results & Conclusion Methods Myocardial infarction (MI) was induced by permanent left anterior descending coronary artery ligation in MerCreMer/ZEG mice. Surviving animals (n=30) with a left ventricular ejection fraction (LVEF) ≤ 45% were treated 3 weeks after MI with either EV (from 1.4 × 106 human iPS-derived CPC; 10 × 109 particles; n=15) or PBS (n=15) delivered by echo-guided intra-myocardial injections targeted to the peri-infarcted area. To track a putative proliferation of endogenous cardiomyocytes, osmotic pumps delivering EdU were implanted for 10-13 days after EV or PBS treatnent. Cardiac function was assessed 4-6 weeks after injections by echocardiography (n=15 p/group) and MRI (n=6-7 p/group), blinded to treatment group. Hearts were harvested and subjected to histological and transcriptomic analyses by qRT-PCR and genome-wide microarrays (Affymetrix). Results In controls, echocardiographically assessed EF slightly decreased over time whereas in the EV-treated group, it increased so that at the end-study time point, the between-group difference in changes from baseline averaged 20.7 % ± 10.5 % (p=0.048). This improvement was confirmed by MRI (11%, p=0.05). EV-treated hearts did not show a different number of new cardiomyocytes (TnT+/EdU+/GFP+) compared to controls (32 analyzed sections/mouse). However, EV-treatment was associated with a reduction in infarct size (-11.9% ± 5.75%), and reduced expression of 4 pro-fibrotic genes (Col1a2, Col3a1, Lox, Col1a2) as assessed by qRT-PCR (n=5-6 p/group). Further, the EV-treatment group showed an over-expression of the anti-fibrotic miRNA 133a-1 (2.13 fold) as compared to controls (n=3 p/group; p=0.0015). Conclusions EV secreted by iPSC-CPC improve cardiac function in chronic heart failure by mechanisms which seem to be largely related to the modulation of fibrosis without participation of a de novo cardiogenesis. Funding Labex Revive, Fondation pour la Recherche Medicale, Fonds Marion Elizabeth BRANCHER

International audience; The present study compared the hemorheological properties between Epo-TAgh mice (a model of erythropoietin deficient mice) and wild-type (WT) control mice. Blood viscosity was determined at several shear rates using a cone-plate viscometer at native and adjusted hematocrit (i.e. 40%). Red blood cell (RBC) deformability was measured by ecktacytometry at several shear stresses and RBC aggregation properties by backscattered technique at adjusted hematocrit (i.e. 40%). Epo-TAgh mice had severe anemia (very low hematocrit), decreased blood viscosity at native hematocrit and slightly reduced RBC deformability at high shear stresses in comparison with WT mice. Blood viscosity at adjusted hematocrit (i.e. 40%) was not different between WT and Epo-TAgh mice. RBC aggregation did not differ between the two mice models. These findings suggest a role of erythropoietin in the regulation of RBC deformability.

ObjectivesTo determine the appropriateness of medical imaging examinations involving radiation and to estimate the effective radiation dose and costs associated.DesignCross-sectional retrospective study.SettingTwo Spanish public tertiary hospitals.Participants2022 medical imaging tests were extracted from the radiology information system in February and March of 2014. MRI and ultrasound examinations were excluded.Primary and secondary outcome measuresFive outcomes were set independently by at least two researchers according to four guidelines: (1) appropriate; (2) inappropriate; (3) inappropriate due to repetition, if the timing to carry out next diagnostic tests was incorrect according to guidelines; (4) not adequately justified, if the referral form did not include enough clinical information to allow us to understand the patient’s clinical condition; and (5) not included in the guidelines, if the referral could not be matched to a clinical scenario described in the guidelines. We estimated the prevalence of the five categories according to relevant clinical and sociodemographic variables and the effective radiation dose and costs for each category.ResultsApproximately half of the imaging tests were deemed as appropriate (967, 47.8%) while one-third (634, 31.4%) were considered inappropriate. 19.6% of the effective dose and 25.2% of the cost were associated with inappropriate tests. Women were less likely than men to have an imaging test classified as appropriate (adjusted OR 0.70,95% CI 0.57 to 0.86). Imaging tests requested by general practitioners were less likely to be considered appropriate than those requested by central services (adjusted OR 0.60, 95% CI 0.38 to 0.93). Mammography and CT were more likely to be appropriate than conventional X-rays.ConclusionThere was a significant frequency of inappropriateness, which resulted in a high percentage of associated effective radiation dose. Percentage of inappropriateness depends on sociodemographic and clinical characteristics such as sex, age, referral physician and medical imaging test.

Los trabajos que se presentan en este volumen, bebiendo y siendo parte del sobresaliente y positivo devenir que acompaña en las últimas décadas al ámbito motriz del ser humano y más concretamente a la parcela de la Educación Física están insertas contextualmente y forman parte de un nuevo proceso renovador que se está dando en el ámbito citado, que va a marcar una nueva etapa en ese devenir y por tanto un salto cualitativo en la interpretación, en el sentido, en la direccionalidad, y en los propios efectos que el movimiento libre y voluntario ejecutado conforme a pautas educativas pueda provocar en las personas.

International audience; The pathophysiology of sporadic Alzheimer's disease (AD) remains uncertain. Along with brain amyloid-β (Aβ) deposits and neurofibrillary tangles, cerebrovascular dysfunction is increasingly recognized as fundamental to the pathogenesis of AD. Using an experimental model of limb ischemia in transgenic APPPS1 mice, a model of AD (AD mice), we showed that microvascular impairment also extends to the peripheral vasculature in AD. At D70 following femoral ligation, we evidenced a significant decrease in cutaneous blood flow (- 29%, P

Clinical trials of cell therapy in stroke favor autologous cell transplantation. To date, feasibility studies have used bone marrow-derived mononuclear cells, but harvesting bone marrow cells is invasive thus complicating bedside treatment. We investigated the therapeutic potential of peripheral blood-derived mononuclear cells (PB-MNC) harvested from diabetic patients and stimulated by ephrin-B2 (PB-MNC+) (500,000 cells), injected intravenously 18–24 hours after induced cerebral ischemia in mice. Infarct volume, neurological deficit, neurogenesis, angiogenesis, and inflammation were investigated as were the potential mechanisms of PB-MNC+ cells in poststroke neurorepair. At D3, infarct volume was reduced by 60% and 49% compared to unstimulated PB-MNC and PBS-treated mice, respectively. Compared to PBS, injection of PB-MNC+ increased cell proliferation in the peri-infarct area and the subventricular zone, decreased microglia/macrophage cell density, and upregulated TGF-βexpression. At D14, microvessel density was decreased and functional recovery was enhanced compared to PBS-treated mice, whereas plasma levels of BDNF, a major regulator of neuroplasticity, were increased in mice treated with PB-MNC+ compared to the other two groups. Cell transcriptional analysis showed that ephrin-B2 induced phenotype switching of PB-MNC by upregulating genes controlling cell proliferation, inflammation, and angiogenesis, as confirmed by adhesion and Matrigel assays.Conclusions. This feasibility study suggests that PB-MNC+ transplantation poststroke could be a promising approach but warrants further investigation. If confirmed, this rapid, noninvasive bedside cell therapy strategy could be applied to stroke patients at the acute phase.