Hypoxic cardiac fibroblasts from failing human hearts decrease cardiomyocyte beating frequency in an ALOX15 dependent manner.

A common denominator for patients with heart failure is the correlation between elevated serum levels of proinflammatory cytokines and adverse clinical outcomes. Furthermore, lipoxygenase-induced inflammation is reportedly involved in the pathology of heart failure. Cardiac fibroblasts, which are abundant in cardiac tissue, are known to be activated by inflammation. We previously showed high expression of the lipoxygenase arachidonate 15 lipoxygenase (ALOX15), which catalyzes the conversion of arachidonic acid to 15-hydroxy eicosatetraenoic acid (15-HETE), in ischemic cardiac tissue. The exact roles of ALOX15 and 15-HETE in the pathogenesis of heart failure are however unknown. Biopsies were collected from all chambers of explanted failing human hearts from heart transplantation patients, as well as from the left ventricles from organ donors not suffering from chronic heart failure. Biopsies from the left ventricles underwent quantitative immunohistochemical analysis for ALOX15/B. Gene expression of ALOX enzymes, as well as 15-HETE levels, were examined in cardiac fibroblasts which had been cultured in either hypoxic or normoxic conditions after isolation from failing hearts. After the addition of fibroblast supernatants to human induced pluripotent stem cell-derived cardiomyocytes, intracellular calcium concentrations were measured to examine the effect of paracrine signaling on cardiomyocyte beating frequency. While ALOX15 and ALOX15B were expressed throughout failing hearts as well as in hearts from organ donors, ALOX15 was expressed at significantly higher levels in donor hearts. Hypoxia resulted in a significant increase in gene and protein expression of ALOX15 and ALOX15B in fibroblasts isolated from the different chambers of failing hearts. Finally, preconditioned medium from hypoxic fibroblasts decreased the beating frequency of human cardiomyocytes derived from induced pluripotent stem cells in an ALOX15-dependent manner. In summary, our results demonstrate that ALOX15/B signaling by hypoxic cardiac fibroblasts may play an important role in ischemic cardiomyopathy, by decreasing cardiomyocyte beating frequency

Human intracardiac SSEA4+CD34 cells show features of cycling, immature cardiomyocytes and are distinct from Side Population and C-kit+CD45- cells

Cardiomyocyte proliferation has emerged as the main source of new cardiomyocytes in the adult. Progenitor cell populations may on the other hand contribute to the renewal of other cell types, including endothelial and smooth muscle cells. The phenotypes of immature cell populations in the adult human heart have not been extensively explored. We therefore investigated whether SSEA4+CD34- cells might constitute immature cycling cardiomyocytes in the adult failing and non-failing human heart. The phenotypes of Side Population (SP) and C-kit+CD45- progenitor cells were also analyzed. Biopsies from the four heart chambers were obtained from patients with end-stage heart failure as well as organ donors without chronic heart failure. Freshly dissociated cells underwent flow cytometric analysis and sorting. SSEA4+CD34- cells expressed high levels of cardiomyocyte, stem cell and proliferation markers. This pattern resembles that of cycling, immature, cardiomyocytes, which may be important in endogenous cardiac regeneration. SSEA4+CD34- cells isolated from failing hearts tended to express lower levels of cardiomyocyte markers as well as higher levels of stem cell markers. C-kit+CD45- and SP CD45- cells expressed high levels of endothelial and stem cell markers-corresponding to endothelial progenitor cells involved in endothelial renewal.CC BY 4.0Copyright:  © 2022 Sandstedt et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.Funding: This study was funded by grants from the Swedish Society of Medicine (JS, https://www.sls.se/); The Gothenburg Society of Medicine (MS,JS, https://goteborgslakaresallskap.se/); the Emelle Foundation (MS); the Heart-Lung Foundation (LMH, https://www.hjart-lungfonden.se/); ALF research grant from the Sahlgrenska University Hospital (JS, LMH, https://www.sahlgrenska.se/); grants from the foundations of the Sahlgrenska University Hospital (MS, JS, https://www.sahlgrenska.se/) and University of Skövde (JSy, https://www.his.se), by grants from the Swedish Knowledge Foundation (https://www.kks.se).There was no additional external fundingreceived for this study. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.</p

The Arachidonate 15-Lipoxygenase Enzyme Product 15-HETE Is Present in Heart Tissue from Patients with Ischemic Heart Disease and Enhances Clot Formation

<div><p>Ischemic heart disease is a major cause of death and morbidity and the search for novel therapeutic targets is still required. We have previously shown that the enzyme arachidonate 15 lipoxygenase (ALOX15), which catalyzes the conversion of arachidonic acid to 15-hydroxy eicosatetraenoic acid (15-HETE), is highly expressed in ischemic heart tissue, but its role in the pathogenesis of ischemic heart disease is unclear. Here we showed that expression of ALOX15, but not ALOX12 or ALOX15B, was increased in ischemic versus non-ischemic human heart biopsy samples. A similar ALOX expression pattern was found in hypoxic human cardiomyocytes and cardiac endothelial cells. We also showed that levels of 15-HETE were significantly higher in ischemic versus non-ischemic human heart biopsy samples and showed a tendency to increase in serum from the patients with ischemic heart disease. Moreover, hypoxia increased the production of 15-HETE levels from human cardiomyocytes and cardiac endothelial cells. The hypoxia-induced increase in 15-HETE levels from human cardiomyocytes was inhibited by the ALOX15 inhibitor baicalein. Finally, by using intrinsic rotational thromboelastometry, we showed that human whole blood clotted faster in the presence of 15-HETE. In summary, we propose that increased ALOX15 expression in heart tissue under ischemic conditions may lead to increased production of 15-HETE, potentially contributing to thrombosis.</p></div

Increased expression of ALOX15 and 15-HETE levels in human ischemic heart biopsies.

<p>(<b>A</b>) ALOX15, ALOX15B and ALOX12 mRNA expression n (relative to the control for their respective gene) in right atrium from control adults (n = 3) and from patients undergoing AVR (n = 5) or CABG (n = 5) surgery. (<b>B</b>) Serial sections of biopsies from right atrium of patients undergoing AVR (left panels) or CABG (right panels) surgery. Sections were stained with DAPI (blue) and antibodies against ALOX15 (green, upper panels) or ALOX15 isotype control (green, lower panels). Scale bar = 50 μm. (<b>C)</b> 15-HETE levels in right atrial tissue samples from patients undergoing AVR (n = 5) or CABG (n = 5) surgery. Data are expressed as mean ± SEM. (<b>D)</b> 15-HETE levels in serum from patients undergoing AVR (n = 5) or CABG (n = 5) surgery. Data are expressed as mean ± SEM. (<b>A</b>) One way ANOVA with Tukey’s multiple comparisons test (** <i>p</i> < 0.01). (<b>C</b>) Mann-Whitney test (** <i>p</i> = 0.0079).</p

Patient characteristics.

<p>Patient characteristics.</p

Inflammatory activation of human cardiac fibroblasts leads to altered calcium signaling, decreased connexin 43 expression and increased glutamate secretion

Cardiac fibroblasts, which are abundant in heart tissue, are involved not only in extracellular matrix homeostasis and repair, but also in cardiac remodeling after a myocardial infarction that, in turn, can lead to loss of cardiac function and heart failure. Ca2+ signaling is functionally important in many cell types, but the roles of fibroblast signaling and inflammation in the pathogenesis of heart disease are unclear. Here, we tested the hypothesis that inflammatory activation affects cardiac fibroblasts, both in terms of Ca2+ signaling and their capacity for intercellular communication through the gap junction channel protein connexin 43 (Cx43). We examined Ca2+ responses induced by known modulators of cardiac function such as glutamate, ATP and 5-hydroxytryptamine (5-HT) in human cardiac fibroblasts, under normal and inflammatory conditions. We showed that activation of human cardiac fibroblasts by lipopolysaccharide (LPS) for 24 h altered Ca2+ signaling, increased TLR4 and decreased Cx43 expression. In the fibroblasts, LPS treatment increased glutamate-evoked and decreased 5-HT-evoked Ca2+ signals. LPS activation also induced increased secretion of glutamate and proinflammatory cytokines from these cells. In summary, we propose that inflammatory stimuli can affect intracellular Ca2+ release, Cx43 expression, glutamate release and cytokine secretion in human cardiac fibroblasts. Inflammatory conditions may, therefore, impair intercellular network communication between fibroblasts and cardiomyocytes potentially contributing to cardiac dysfunction

Increased expression of ALOX15 and 15-HETE levels in hypoxic human cardiac endothelial cells.

<p>(<b>A</b>) ALOX15, ALOX15B and ALOX12 mRNA expression (relative to the control for their respective gene) in human cardiac endothelial cells incubated under normoxic (21% oxygen; control) or hypoxic conditions (1% oxygen) for 6 h (n = 3) (<b>B</b>) Immunocytochemical staining of human cardiac endothelial cells with antibody against ALOX15 (green) and DAPI (blue) in cells incubated in normoxia (control) or hypoxia. Scale bar 40 μm. <b>(C-D)</b> Representative immunoblot and quantification of ALOX15 protein in human cardiac endothelial cells incubated in normoxia (control) or hypoxia. <b>(E)</b> 15-HETE levels in cell culture media from human cardiac endothelial cells incubated in normoxia (control) or hypoxia. Data are expressed as mean ± SEM (<i>n</i> = 3). Student’s t-test (* <i>p</i> < 0.05; ** <i>p</i> < 0.01).</p

Increased expression of ALOX15 and 15-HETE levels in hypoxic human cardiomyocytes.

<p>(<b>A</b>) ALOX15, ALOX15B and ALOX12 mRNA expression (relative to the control for their respective gene) in human cardiomyocytes derived from induced pluripotent stem cells incubated under normoxic (21% oxygen; control) or hypoxic conditions (1% oxygen) for 6 h (n = 3). <b>(B)</b> Immunocytochemical staining of human cardiomyocytes with antibody against ALOX15 (green) and DAPI (blue) in cells incubated in normoxia (control) or hypoxia. Scale bar = 40 μm. <b>(C-D)</b> Representative immunoblot and quantification of ALOX15 protein in human cardiomyocytes incubated in normoxia (control) or hypoxia (n = 3), <b>(E)</b> 15-HETE levels in cell culture media from cardiomyocytes incubated in normoxia (control) or hypoxia. <b>(F)</b> 15-HETE levels in lysates from human cardiomyocytes incubated in normoxia (control) or hypoxia with or without baicalein (25 μmol/L) for 6 h (n = 3–6). Data are expressed as mean ± SEM. (<b>A-D</b>) Student’s t-test (* <i>p</i> < 0.05; ** <i>p</i> < 0.01). (<b>F</b>) One-way ANOVA with Tukey's multiple comparisons test (*** <i>p</i> < 0.001).</p

Incidence and Survival Analyses in Children with Solid Tumours Diagnosed in Sweden 1983-2007.

Aim: Solid tumours constitute 40% of childhood malignancies. The Swedish Childhood Cancer Registry is population-based and includes all children with cancer reported from the six paediatric oncology centres in Sweden. The aim was to investigate incidence and survival. Methods: We used the new WHO ICCC-3 for reclassification of the patients. Incidence and survival analyses were performed in the study population. Results: 2 487 children (< 15 years) were diagnosed with solid tumours in Sweden 1983 - 2007. The distribution of diagnoses was similar to that reported in other studies. The annual incidence was 65.3/million children. The survival rates at 10 years follow up have improved significantly when comparing the two time periods 1983-95 and 1995-2007 (76 vs. 82%; p<0.01). Conclusions: The mean annual incidence of solid tumours in children was 65.3/ million and has been stable during the study period. Survival rates for solid tumours at 5, 10 and 20 years follow up were 80, 79 and 76%, respectively