Highlights d Cardiac fibroblasts and endothelial cells induce hiPSCcardiomyocyte maturation d CX43 gap junctions form between cardiac fibroblasts and cardiomyocytes d cAMP-pathway activation contributes to hiPSCcardiomyocyte maturation d Patient-derived hiPSC-cardiac fibroblasts cause arrhythmia in microtissues

Human endothelial cells (ECs) and pericytes are of great interest for research on vascular development and disease, as well as for future therapy. This protocol describes the efficient generation of ECs and pericytes from human pluripotent stem cells (hPSCs) under defined conditions. Essential steps for hPSC culture, differentiation, isolation and functional characterization of ECs and pericytes are described. Substantial numbers of both cell types can be derived in only 2-3 weeks: this involves differentiation (10 d), isolation (1 d) and 4 or 10 d of expansion of ECs and pericytes, respectively. We also describe two assays for functional evaluation of hPSC-derived ECs: (i) primary vascular plexus formation upon coculture with hPSC-derived pericytes and (ii) incorporation in the vasculature of zebrafish xenografts in vivo. These assays can be used to test the quality and drug sensitivity of hPSC-derived ECs and model vascular diseases with patient-derived hPSCs.

High‐mobility group box 1 (HMGB1) is released extracellularly upon cell necrosis acting as a mediator in tissue injury and inflammation. However, the molecular mechanisms for the proinflammatory effect of HMGB1 are poorly understood. Here, we define a novel function of HMGB1 in promoting Mac‐1‐dependent neutrophil recruitment. HMGB1 administration induced rapid neutrophil recruitment in vivo. HMGB1‐mediated recruitment was prevented in mice deficient in the β2‐integrin Mac‐1 but not in those deficient in LFA‐1. As observed by bone marrow chimera experiments, Mac‐1‐dependent neutrophil recruitment induced by HMGB1 required the presence of receptor for advanced glycation end products (RAGE) on neutrophils but not on endothelial cells. In vitro, HMGB1 enhanced the interaction between Mac‐1 and RAGE. Consistently, HMGB1 activated Mac‐1 as well as Mac‐1‐mediated adhesive and migratory functions of neutrophils in a RAGE‐dependent manner. Moreover, HMGB1‐induced activation of nuclear factor‐κB in neutrophils required both Mac‐1 and RAGE. Together, a novel HMGB1‐dependent pathway for inflammatory cell recruitment and activation that requires the functional interplay between Mac‐1 and RAGE is described here.

Cardiomyocytes and endothelial cells in the heart are in close proximity and in constant dialogue. Endothelium regulates the size of the heart, supplies oxygen to the myocardium and secretes factors that support cardiomyocyte function. Robust and predictive cardiac disease models that faithfully recapitulate native human physiology in vitro would therefore ideally incorporate this cardiomyocyte-endothelium crosstalk. Here, we have generated and characterized human cardiac microtissues in vitro that integrate both cell types in complex 3D structures. We established conditions for simultaneous differentiation of cardiomyocytes and endothelial cells from human pluripotent stem cells following initial cardiac mesoderm induction. The endothelial cells expressed cardiac markers that were also present in primary cardiac microvasculature, suggesting cardiac endothelium identity. These cell populations were further enriched based on surface markers expression, then recombined allowing development of beating 3D structures termed cardiac microtissues. This in vitro model was robustly reproducible in both embryonic and induced pluripotent stem cells. It thus represents an advanced human stem cell-based platform for cardiovascular disease modelling and testing of relevant drugs.

Human neutrophil-specific CD177 (NB1 and PRV-1) has been reported to be up-regulated in a number of inflammatory settings, including bacterial infection and granulocyte-colonystimulating factor application. Little is known about its function. By flow cytometry and immunoprecipitation studies, we identified platelet endothelial cell adhesion molecule-1 (PECAM-1) as a binding partner of CD177. Real-time proteinprotein analysis using surface plasmon resonance confirmed a cation-dependent, specific interaction between CD177 and the heterophilic domains of PECAM-1. Monoclonal antibodies against CD177 and against PECAM-1 domain 6 inhibited adhesion of U937 cells stably expressing CD177 to immobilized PECAM-1. Transendothelial migration of human neutrophils was also inhibited by these antibodies. Our findings provide direct evidence that neutrophil-specific CD177 is a heterophilic binding partner of PECAM-1. This interaction may constitute a new pathway that participates in neutrophil transmigration.CD177 (NB1 and PRV-1) is a 58-to 64-kDa glycosylphosphatidylinositol-anchored glycoprotein expressed exclusively by neutrophils, neutrophilic metamyelocytes, and myelocytes, but not by any other blood cells (1, 2). We and others elucidated its primary structure by sequencing the NB1 and PRV-1 genes, which later turned out to be two alleles of a single CD177 gene (3-5). The surface expression of CD177 is unique in that only a subpopulation of neutrophils expresses this protein on the cell surface, with the mean size of the CD177-positive subpopulation ranging from 45% to 65% (2, 6).CD177 has been well studied as a target antigen in immunemediated disorders. During pregnancy, women with a CD177 null phenotype are prone to produce alloantibodies against CD177 that cross the placenta, react with fetal neutrophils, and cause neutropenia of the newborn. This mechanism let to the initial discovery of the NB1 antigen in 1971 (7). Alloantibodies to CD177, present in blood products obtained from immunized donors, have also been implicated as mediators of transfusionrelated acute lung injury (8).Although well characterized as an immunotarget, the function of CD177 is largely unknown. It has been reported that CD177 is up-regulated on the neutrophil surface upon stimulation, including during severe bacterial infections, and following granulocyte-colony-stimulating factor treatment (9). In addition, antibody-mediated clustering of CD177 primes the N-formyl-methionyl-leucyl-phenylalanine (fMLP) 3 -activated respiratory burst reaction of the neutrophil (8). Taken together, these observations make it reasonable to suppose that CD177 may be involved in processes of neutrophil-mediated host defense. One preliminary study suggests a participation of CD177 in neutrophil-endothelial cell interaction (10). The latter observation is in line with the fact that CD177, as a member of the leukocyte antigen-6 superfamily, shares a similar structure with the urokinase plasminogen activator receptor (11). Urokinase plasminogen activator receptor is expr...

Objective-Endothelial cells (ECs), pericytes, and vascular smooth muscle cells (vSMCs)

Mammalian ALDH7A1 is homologous to plant ALDH7B1, an enzyme that protects against various forms of stress, such as salinity, dehydration, and osmotic stress. It is known that mutations in the human ALDH7A1 gene cause pyridoxine-dependent and folic acid-responsive seizures. Herein, we show for the first time that human ALDH7A1 protects against hyperosmotic stress by generating osmolytes and metabolizing toxic aldehydes. Human ALDH7A1 expression in Chinese hamster ovary cells attenuated osmotic stress-induced apoptosis caused by increased extracellular concentrations of sucrose or sodium chloride. Purified recombinant ALDH7A1 efficiently metabolized a number of aldehyde substrates, including the osmolyte precursor, betaine aldehyde, lipid peroxidation-derived aldehydes, and the intermediate lysine degradation product, ␣-aminoadipic semialdehyde. The crystal structure for ALDH7A1 supports the enzyme's substrate specificities. Tissue distribution studies in mice showed the highest expression of ALDH7A1 protein in liver, kidney, and brain, followed by pancreas and testes. ALDH7A1 protein was found in the cytosol, nucleus, and mitochondria, making it unique among the aldehyde dehydrogenase enzymes. Analysis of human and mouse cDNA sequences revealed mitochondrial and cytosolic transcripts that are differentially expressed in a tissue-specific manner in mice. In conclusion, ALDH7A1 is a novel aldehyde dehydrogenase expressed in multiple subcellular compartments that protects against hyperosmotic stress by generating osmolytes and metabolizing toxic aldehydes. The human aldehyde dehydrogenase (ALDH)3 superfamily contains 19 enzymes involved in the NAD(P)ϩ -dependent oxidation of aldehydes to their corresponding carboxylic acids. These enzymes play crucial roles in a number of physiological processes by efficiently metabolizing a wide array of endogenous and exogenous aldehydes (1). Aldehydes are highly reactive molecules that can form adducts resulting in DNA damage and enzyme inactivation. As such, their removal is of the utmost importance. ALDH enzymes also couple the removal of these potentially toxic aldehydes to NAD(P)H production, which, in turn, helps maintain cellular redox balance. Finally, ALDH activity generates a number of important cellular molecules, including retinoic acid, the neurotransmitter ␥-aminobutyric acid, the major dietary folate tetrahydrofolate, and the osmolyte betaine (1).Human ALDH7A1 was originally identified as sharing 60% homology with the osmotic stress-induced 26g pea turgor protein (according to the official nomenclature now referred to as ALDH7B1), found in the common garden pea (Pisum sativum) (2). Initially named "antiquitin," the ALDH7A1 protein has been highly conserved throughout evolution. Indeed, the degree of homology noted between ALDH7A1 and ALDH7B1 is comparable with that observed between the human and pea histone H2A proteins, which are among the most evolutionarily conserved of all eukaryotic proteins (2, 3). Such a high degree of sequence similarity between species ofte...

The anthracycline doxorubicin (Doxo) and its analogs daunorubicin (Daun), epirubicin (Epi), and idarubicin (Ida) have been cornerstones of anticancer therapy for nearly five decades. However, their clinical application is limited by severe side effects, especially dose-dependent irreversible cardiotoxicity. Other detrimental side effects of anthracyclines include therapy-related malignancies and infertility. It is unclear whether these side effects are coupled to the chemotherapeutic efficacy. Doxo, Daun, Epi, and Ida execute two cellular activities: DNA damage, causing double-strand breaks (DSBs) following poisoning of topoisomerase II (Topo II), and chromatin damage, mediated through histone eviction at selected sites in the genome. Here we report that anthracycline-induced cardiotoxicity requires the combination of both cellular activities. Topo II poisons with either one of the activities fail to induce cardiotoxicity in mice and human cardiac microtissues, as observed for aclarubicin (Acla) and etoposide (Etop). Further, we show that Doxo can be detoxified by chemically separating these two activities. Anthracycline variants that induce chromatin damage without causing DSBs maintain similar anticancer potency in cell lines, mice, and human acute myeloid leukemia patients, implying that chromatin damage constitutes a major cytotoxic mechanism of anthracyclines. With these anthracyclines abstained from cardiotoxicity and therapy-related tumors, we thus uncoupled the side effects from anticancer efficacy. These results suggest that anthracycline variants acting primarily via chromatin damage may allow prolonged treatment of cancer patients and will improve the quality of life of cancer survivors.