Human CD16+ monocytes promote a pro-atherosclerotic endothelial cell phenotype via CX3CR1–CX3CL1 interaction

Aims Monocytes are central for atherosclerotic vascular inflammation. The human non-classical, patrolling subtype, which expresses high levels of CD16 and fractalkine receptor CX3CR1, strongly associates with cardiovascular events. This is most marked in renal failure, a condition with excess atherosclerosis morbidity. The underlying mechanism is not understood. This study investigated how human CD16+ monocytes modulate endothelial cell function. Methods and results In patients with kidney failure, CD16+ monocyte counts were elevated and dynamically decreased within a year after transplantation, chiefly due to a drop in CD14+CD16+ cells. The CX3CR1 ligand CX3CL1 was similarly elevated in the circulation of humans and mice with renal impairment. CX3CL1 up-regulation was also observed close to macrophage rich human coronary artery plaques. To investigate a mechanistic basis of this association, CD16+CX3CR1HIGH monocytes were co-incubated with primary human endothelium in vitro. Compared to classical CD14+ monocytes or transwell cocultures, CD16+ monocytes enhanced endothelial STAT1 and NF-κB p65 phosphorylation, up-regulated expression of CX3CL1 and interleukin-1β, numerous CCL and CXCL chemokines and molecules promoting leucocyte patrolling and adhesion such as ICAM1 and VCAM1. Genes required for vasodilatation including endothelial nitric oxide synthase decreased while endothelial collagen production increased. Uraemic patients’ monocytes enhanced endothelial CX3CL1 even more markedly. Their receptor CX3CR1 was required for enhanced aortic endothelial stiffness in murine atherosclerosis with renal impairment. CX3CR1 dose-dependently modulated monocyte-contact-dependent gene expression in human endothelium. Conclusion By demonstrating endothelial proatherosclerotic gene regulation in direct contact with CD16+ monocytes, in part via cellular CX3CR1–CX3CL1 interaction, our data delineate a mechanism how this celltype can increase cardiovascular risk.

Association of N6-methyladenine DNA with plaque progression in atherosclerosis via myocardial infarction-associated transcripts

Modification of the novel N6-methyladenine (m6A) DNA implicates this epigenetic mark in human malignant disease, but its role in atherosclerosis (AS) is largely unknown. Here, we found that the leukocyte level of m6A but not 5mC DNA modification was decreased with increasing of carotid plaque size and thickness in 207 AS patients as compared with 142 sex- and age-matched controls. Serum low-density lipoprotein (LDL) and leukocyte m6A levels were associated with the progression of carotid plaque size and thickness. Both LDL level and plaque thickness were also independently and negatively related to m6A level. Reduced m6A level was further confirmed in leukocytes and endothelium in western diet-induced AS mice and in oxidized-LDL (ox-LDL)-treated human endothelium and monocyte cells. Decreased m6A level was closely related to the upregulation of AlkB homolog 1 (ALKBH1), the demethylase of m6A. Silencing of ALKBH1 or hypoxia-inducible factor 1α (HIF1α) could rescue the ox-LDL–increased level of MIAT, a hypoxia-response gene. Mechanically, ox-LDL induced HIF1α for transfer into the nucleus. Nuclear HIF1α bound to the ALKBH1-demethylated MIAT promoter and transcriptionally upregulated its expression. Therefore, elevated ALKBH1 level in endothelium and leukocytes reduced m6A level, which is a novel and sensitive biomarker for AS progression.

Reprogrammed Adult Human Endothelium into Hematopoietic Stem Cells Yields Functional T Cells In Vivo

During development, the hematopoietic stem cells that go on to populate the bone marrow and give rise to all blood cell lineages emerge from a specialized endothelial subpopulation. We have previously harnessed this vestigial identity to achieve the direct conversion of adult endothelial cells (ECs) into long-term engraftable hematopoietic stem and progenitor cells (rEC-HSPCs); however, to date, we had only detected and characterized functional T cells that result from the transplantation, engraftment, and differentiation of mouse rEC-HSPCs (Lis, R. et al., Conversion of adult endothelium to immunocompetent haematopoietic stem cells. Nature, 545:439-445. 2017). We first reprogrammed adult human endothelium using lentiviral vectors encoding FOSB, GFI1, SPI1, and RUNX1 (FGRS) under constitutive promoters and an inductive vascular niche (Sandler V. et al., Reprogramming of human endothelium into hematopoietic cells requires vascular niche induction. Nature, 511:312-8. 2014). The resulting rEC-HSPCs maintained exogenous expression of the four transcription factors for over 20 weeks post-transplantation into immuno-compromised NSG mice. Constitutive expression of Spi1, however, has been shown to hinder lymphoid differentiation in vivo by blocking T lymphopoiesis (Anderson, M. et al. Constitutive expression of PU.1 in fetal hematopoietic progenitors blocks T cell development at the pro-T cell stage. Immunity 16:285-296. 2002). In addition, mice of the NSG strain cannot educate native B or T cells to maturity. Our system of constitutive exogenous FGRS expression was therefore unable to confer transplanted immuno-compromised mice the ability to generate an adaptive immune response. Here, we obtained human rEC-HSPCs making use of (i) doxycycline-inducible vectors to temporarily overexpress FGRS and (ii) transgenic substrains of NSG mice (one carrying human stem cell factor, granulocyte/macrophage colony-stimulating factor, and interleukin 3; the other, human major histocompatibility complex class I as well as beta-2 microglobulin) for transplantation assays. We show that human rEC-HSPCs engraft primary- and secondary-transplanted mice for over a year at levels of up to 20% in the spleen or bone marrow. Engrafted cells differentiate into all blood lineages including phenotypically and functionally mature T cells in the absence of exogenous FGRS expression in vivo. Notably, the resulting T cells undergo TCR rearrangement and are able to clear viral particles one week post-LCMV infection. Functional and phenotypic analyses are presented in juxtaposition with experiments using cord blood-transplanted mice. These results demonstrate that our present direct conversion strategy generates bona fide human hematopoietic stem cells from adult endothelial cells. Disclosures Rafii: Angiocrine Bioscience: Equity Ownership.

Influence of long-term treatment with glyceryl trinitrate on remote ischemic conditioning

Remote ischemic conditioning (RIC) protects against sustained myocardial ischemia. Because of overlapping mechanisms, this protection may be altered by glyceryl trinitrate (GTN), which is commonly used in the treatment of patients with chronic ischemic heart disease. We investigated whether long-term GTN treatment modifies the protection by RIC in the rat myocardium and human endothelium. We studied infarct size (IS) in rat hearts subjected to global ischemia-reperfusion (I/R) in vitro and endothelial function in healthy volunteers subjected to I/R of the upper arm. In addition to allocated treatment, rats were coadministered with reactive oxygen species (ROS) or nitric oxide (NO) scavengers. Rats and humans were randomized to 1) control, 2) RIC, 3) GTN, and 4) GTN + RIC. In protocols 3 and 4, rats and humans underwent long-term GTN treatment for 7 consecutive days, applied subcutaneously or 2 h daily transdermally. In rats, RIC and long-term GTN treatment reduced mean IS (18 ± 12%, P = 0.007 and 15 ± 5%, P = 0.002) compared with control (35 ± 13%). RIC and long-term GTN treatment in combination did not reduce IS (29 ± 12%, P = 0.55 vs. control). ROS and NO scavengers both attenuated IS reduction by RIC and long-term GTN treatment. In humans, I/R reduced endothelial function ( P = 0.01 vs. baseline). Separately, RIC and long-term GTN prevented the reduction in endothelial function caused by I/R; given in combination, prevention was lost. RIC and long-term GTN treatment both protect against rat myocardial and human endothelial I/R injury through ROS and NO-dependent mechanisms. However, when given in combination, RIC and long-term GTN treatment fail to confer protection. NEW & NOTEWORTHY Remote ischemic conditioning (RIC) and long-term glyceryl trinitrate (GTN) treatment protect against ischemia-reperfusion injury in both human endothelium and rat myocardium. However, combined application of RIC and long-term GTN treatment abolishes the individual protective effects of RIC and GTN treatment on ischemia-reperfusion injury, suggesting an interaction of clinical importance.