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Haematopoietic stem cells (HSCs) are generated from haemogenic endothelial (HE) cells via the formation of intra-aortic haematopoietic clusters (IAHCs) in vertebrate embryos. The molecular events controlling endothelial specification, endothelial-to-haematopoietic transition (EHT) and IAHC formation, as it occurs in vivo inside the aorta, are still poorly understood. To gain insight in these processes, we performed single-cell RNA-sequencing of non-HE cells, HE cells, cells undergoing EHT, IAHC cells, and whole IAHCs isolated from mouse embryo aortas. Our analysis identified the genes and transcription factor networks activated during the endothelial-to-haematopoietic switch and IAHC cell maturation toward an HSC fate. Our study provides an unprecedented complete resource to study in depth HSC generation in vivo. It will pave the way for improving HSC production in vitro to address the growing need for tailor-made HSCs to treat patients with blood-related disorders., Haematopoietic stem cells (HSCs) are generated from haemogenic endothelial (HE) cells in vertebrate embryo aortas. Here, the authors perform single-cell RNA-sequencing of cells isolated from embryonic mouse aortas to identify genes and transcription factor networks activated during the endothelial-to-haematopoietic switch.

BACKGROUND/AIMS Smooth muscle tone is controlled by Ca(2+) signaling in the endothelial layer. Mouse endothelial cells are interconnected by gap junctions made of Connexin40 (Cx40) and Cx37 which allow the exchange of signaling molecules to coordinate their activity. Here we investigated the role of Cx40 in the endothelial Ca(2+) signaling of the mouse aorta. METHODS Ca(2+) imaging was performed on intact aortic endothelium from both wild type (Cx40+/+) and Connexin40 deficient (Cx40 / ) mice. RESULTS Acetylcholine (ACh) induced early fast and high amplitude Ca(2+) transients in a fraction of endothelial cells expressing the M3 muscarinic receptors. Inhibition of intercellular communication using carbenoxolone or octanol fully blocked the propagation of ACh induced Ca(2+) transients toward adjacent cells in WT and Cx40 / mice. As compared to WT Cx40 / mice displayed a reduced propagation of ACh induced Ca(2+) waves indicating that Cx40 contributes to the spreading of Ca(2+) signals. The propagation of those Ca(2+) responses was not blocked by suramin a blocker of purinergic ATP receptors indicating that there is no paracrine effect of ATP release on the Ca(2+) waves. CONCLUSIONS Altogether our data show that Cx40 and Cx37 contribute to the propagation and amplification of the Ca(2+) signaling triggered by ACh in endothelial cells expressing the M3 muscarinic receptors.

BACKGROUND Anti Gala13Galß R natural antibodies are responsible for hyperacute rejection in pig to primate xenotransplantation. Although the generation of pigs lacking the a13galactosyltransferase (GalT) has overcome hyperacute rejection antibody mediated rejection is still a problem. It is possible that other enzymes synthesize antigens similar to Gala13Gal epitopes that are recognized by xenoreactive antibodies. The glycosphingolipid isoglobotrihexosylceramide (iGb3) represents such a candidate expressing an alternative Gala13Gal epitope. The present work determined whether the terminal Gala13Gal disaccharide is completely absent in Immerge pigs lacking the GalT using several different highly sensitive methods. METHODS The expression of Gala13Gal was evaluated using a panel of antibodies and lectins by flow cytometry and fluorescent microscopy; GalT activity was detected by an enzymatic assay; and ion trap mass spectroscopy of neutral cellular membranes extracted from aortic endothelial was used for the detection of sugar structures. Finally the presence of iGb3 synthase mRNA was tested by RT PCR in pig thymus spleen lymph node kidney lung and liver tissue samples. RESULTS Aortic endothelial cells derived from GalT knockout pigs expressed neither Gala13Gal nor iGb3 on their surface and GalT enzymatic activity was also absent. Lectin staining showed an increase in the blood group H type sugar structures present in GalT knockout cells as compared to wild type pig aortic endothelial cells (PAEC). Mass spectroscopic analysis did not reveal Gala13Gal in membranes of GalT knockout PAEC; iGb3 was also totally absent whereas a fucosylated form of iGb3 was detected at low levels in both pig aortic endothelial cell extracts. Isoglobotrihexosylceramide 3 synthase mRNA was expressed in all pig tissues tested whether derived from wild type or GalT knockout animals. CONCLUSIONS These results confirm unequivocally the absence of terminal Gala13Gal disaccharides in GalT knockout endothelial cells. Future work will have to focus on other mechanisms responsible for xenograft rejection in particular non Gala13Gal antibodies and cellular responses.

BACKGROUND: Natural killer (NK) cells participate in the immune response against solid organ allo- and xenografts and are tightly regulated through signals mediated by inhibiting and activating receptors expressed on their cell surface. Human NK cytotoxicity against porcine endothelial cells (pEC) is mediated by the interaction of the activating human NK receptor hNKG2D and its corresponding ligand on pEC, porcine UL-16 binding protein 1 (pULBP1). The aim of the present study was to characterize the regulation of pULBP1 cell-surface expression on primary porcine aortic endothelial cells (PAEC). METHODS: A monoclonal antibody (mAb; aE5-63) directed against pULBP1 was generated by immunizing C57BL/6 mice with the pEC line PEDSV.15, and used in cellular ELISA to determine pULBP1 cell surface expression. PAEC were either left untreated or stimulated with human or porcine cytokines [interferon gamma (IFN-gamma), tumor necrosis factor alpha (TNF-alpha)], human serum, cultured under hypoxic conditions, or infected with human or porcine cytomegalovirus (CMV). RESULTS: Neither human nor porcine IFN-gamma stimulation changed pULBP1 expression, whereas both human and porcine TNF-alpha stimulation as well as human and porcine CMV infection significantly decreased pULBP1 expression on PAEC. Coculture of PAEC with human serum strongly increased pULBP1 expression depending on the binding of human anti-pig antibodies. Exposure of PAEC to hypoxia only slightly increased pULBP1 expression. CONCLUSIONS: In conclusion, (i) the novel anti-pULBP1 IgM mAb aE5-63 represents a useful tool to study pULBP1/hNKG2D-mediated responses in xenotransplantation, and (ii) the expression of pULBP1, a human-pig cross-species functional hNKG2D ligand, on the surface of PAEC is modulated by various stimuli associated with transplantation.

Objectives— Basic fibroblast growth factor (bFGF) stimulates vascular smooth muscle cell (SMC) migration. We determined whether bFGF increases SMC reactive oxygen-species (ROS) and studied the role of ROS for SMC migration. Methods and Results— bFGF rapidly increased rat SMC ROS formation and migration through pathways sensitive to inhibition of NADPH oxidases, PI3-kinase, protein kinase C, and Rac-1. SiRNA directed against the NADPH oxidase Nox4 impaired basal but not bFGF-induced ROS formation and did not affect migration. In contrast, siRNA against Nox1 blocked the agonist-induced ROS generation as well as the bFGF-induced migration. Agonist-induced migration was also attenuated in SMC derived from Nox1 y/− mice and transduction of Nox1 restored normal migration. Likewise, SMC outgrowth in response to bFGF was attenuated in aortic segments from Nox1 y/− mice as compared with Nox1 y/+ mice. bFGF activated JNK but not Src in a Nox1-dependent manner. Consequently, phosphorylation of the adaptor protein paxillin, which is central for migration and secretion of matrix-metalloproteinases, were dependent on Nox1 as well as JNK but not Src. Conclusions— These data demonstrate that bFGF activates the Nox1-containing NADPH oxidase and that bFGF through a pathway involving ROS and JNK stimulates SMC migration.

Background Connexin37 (Cx37) and Cx40 are crucial for endothelial cell-cell communication and homeostasis. Both connexins interact with endothelial nitric oxide synthase (eNOS). The exact contribution of these interactions to the regulation of vascular tone is unknown. Results Cx37 and Cx40 were expressed in close proximity to eNOS at cell-cell interfaces of mouse aortic endothelial cells. Absence of Cx37 did not affect expression of Cx40 and a 50 % reduction of Cx40 in Cx40+/− aortas did not affect the expression of Cx37. However, absence of Cx40 was associated with reduced expression of Cx37. Basal NO release and the sensitivity for ACh were decreased in Cx37−/− and Cx40−/− aortas but not in Cx40+/− aortas. Moreover, ACh-induced release of constricting cyclooxygenase products was present in WT, Cx40−/− and Cx40+/− aortas but not in Cx37−/− aortas. Finally, agonist-induced NO-dependent relaxations and the sensitivity for exogenous NO were not affected by genotype. Conclusions Cx37 is more markedly involved in basal NO release, release of cyclooxygenase products and the regulation of the sensitivity for ACh as compared to Cx40. Electronic supplementary material The online version of this article (doi:10.1186/s12964-015-0110-1) contains supplementary material, which is available to authorized users.

Abstract —We compared the effects of NO donors and cGMP analogues on the growth of aortic smooth muscle cells (SMCs) derived from newborn, adult (aged 3 months), and old (aged 2 years) rats. We found that the NO donor S -nitroso- N -acetylpenicillamine failed to block DNA synthesis in SMCs from old rats but was effective in SMCs from newborn and adult rats. However, cGMP analogues were inhibitory in all 3 SMC types. We demonstrated that in SMCs from old rats, NO was unable to increase the concentration of intracellular cGMP, suggesting that either cGMP synthesis was defective or cGMP degradation was enhanced. Western blot analysis revealed that SMCs from old rats do not express the β subunit of soluble guanylyl cyclase. To confirm the importance of this observation in vivo, we balloon-injured the carotid arteries of adult and old rats. Whereas soluble guanylyl cyclase was expressed at the same level in the media of injured vessels and uninjured vessels of both groups, its expression in the intimas of old rats was reduced by 70% compared with intimas from adult animals. Furthermore, N ω -nitro- l -arginine, an inhibitor of NO synthesis, enhanced the intimal thickening in injured vessels in adult rats but not in old rats. We conclude that the loss of NO responsiveness in aged rats is due to the lack of the β subunit of soluble guanylyl cyclase, and we speculate that this defect contributes to the enhanced intimal thickening in response to injury in old animals.