Endothelial cells are integral components of all vasculature within complex organisms. As they line the blood vessel wall, endothelial cells are constantly exposed to a variety of molecular factors and shear force that can induce cellular damage and stress. However, how endothelial cells are removed or eliminate unwanted cellular contents, remains unclear. The generation of large extracellular vesicles (EVs) has emerged as a key mechanism for the removal of cellular waste from cells that are dying or stressed. Here, we used intravital microscopy of the bone marrow to directly measure the kinetics of EV formation from endothelial cells in vivo under homoeostatic and malignant conditions. These large EVs are mitochondria-rich, expose the 'eat me' signal phosphatidylserine, and can interact with immune cell populations as a potential clearance mechanism. Elevated levels of circulating EVs correlates with degradation of the bone marrow vasculature caused by acute myeloid leukaemia. Together, our study provides in vivo spatio-temporal characterization of EV formation in the murine vasculature and suggests that circulating, large endothelial cell-derived EVs can provide a snapshot of vascular damage at distal sites.
© 2024. The Author(s).

Endothelial progenitor cell (EPC) dysfunction contributes to vascular disease in diabetes mellitus. However, the molecular mechanism underlying EPC dysfunction and its contribution to delayed reendothelialization in diabetes mellitus remain unclear. Our study aimed to illustrate the potential molecular mechanism underlying diabetic EPC dysfunction in vivo and in vitro. Furthermore, we assessed the effect of EPC transplantation on endothelial regeneration in diabetic rats.
Late outgrowth EPCs were isolated from the bone marrow of rats for in vivo and in vitro studies. In vitro functional assays and Western blotting were conducted to reveal the association between C-X-C chemokine receptor type 7 (CXCR7) expression and diabetic EPC dysfunction. To confirm the association between cellular CXCR7 levels and EPC function, CXCR7 expression in EPCs was upregulated and downregulated via lentiviral transduction and RNA interference, respectively. Western blotting was used to reveal the potential molecular mechanism by which the Stromal-Derived Factor-1 (SDF-1)/CXCR7 axis regulates EPC function. To elucidate the role of the SDF-1/CXCR7 axis in EPC-mediated endothelial regeneration, a carotid artery injury model was established in diabetic rats. After the model was established, saline-treated, diabetic, normal, or CXCR7-primed EPCs were injected via the tail vein.
Diabetic EPC dysfunction was associated with decreased CXCR7 expression. Furthermore, EPC dysfunction was mimicked by knockdown of CXCR7 in normal EPCs. However, upregulating CXCR7 expression reversed the dysfunction of diabetic EPCs. The SDF-1/CXCR7 axis positively regulated EPC function by activating the AKT-associated Kelch-like ECH-associated protein 1 (keap-1)/nuclear factor erythroid 2-related factor 2 (Nrf2) axis, which was reversed by blockade of AKT and Nrf2. Transplantation of CXCR7-EPCs accelerated endothelial repair and attenuated neointimal hyperplasia in diabetes mellitus more significantly than transplantation of diabetic or normal EPCs. However, the therapeutic effect of CXCR7-EPC transplantation on endothelial regeneration was reversed by knockdown of Nrf2 expression.
Dysfunction of diabetic EPCs is associated with decreased CXCR7 expression. Furthermore, the SDF-1/CXCR7 axis positively regulates EPC function by activating the AKT/keap-1/Nrf2 axis. CXCR7-primed EPCs might be useful for endothelial regeneration in diabetes-associated vascular disease.

Endothelial progenitor cell (EPC) dysfunction impairs vascular function and remodeling in inflammation-associated diseases, including preeclampsia. However, the underlying mechanism of this inflammation-induced dysfunction remains unclear. In the present study, we found increases in TNF-α and miR-31/155 levels and reduced numbers of circulating EPCs in patients with preeclampsia. Patient-derived mononuclear cells (MNCs) cultured in autologous serum had decreased endothelial nitric oxide synthase (eNOS) expression, nitric oxide production, and differentiation into EPCs with angiogenic potential, and these effects were inhibited by a TNF-α-neutralizing antibody and miR-31/155 inhibitors. Moreover, TNF-α treatment of normal MNCs increased miR-31/155 biogenesis, decreased eNOS expression, reduced EPC differentiation, and impaired angiogenic potential. The TNF-α-induced impairment of EPC differentiation and function was rescued by NF-κB p65 knockdown or miR-31/155 inhibitors. In addition, treatment of MNCs with synthetic miR-31/155 or an eNOS inhibitor mimicked the inhibitory effects of TNF-α on eNOS expression and EPC functions. Moreover, transplantation of EPCs that had been differentiated from TNF-α-treated MNCs decreased neovascularization and blood perfusion in ischemic mouse hindlimbs compared with those of normally differentiated EPCs. These findings suggest that NF-κB activation is required for TNF-α-induced impairment of EPC mobilization, differentiation, and function via miR-31/155 biogenesis and eNOS downregulation. Our data provide a new role for NF-κB-dependent miR-31/155 in EPC dysfunction under the pathogenic conditions of inflammation-associated vascular diseases, including preeclampsia.

Primary hypertension is a major risk factor for ischemic heart disease, stroke, and chronic kidney disease. Insights obtained from the study of rare Mendelian forms of hypertension have been invaluable in elucidating the mechanisms causing primary hypertension and development of antihypertensive therapies. Endothelial cells play a key role in the regulation of blood pressure; however, a Mendelian form of hypertension that is primarily due to endothelial dysfunction has not yet been described. Here, we show that the urea cycle disorder, argininosuccinate lyase deficiency (ASLD), can manifest as a Mendelian form of endothelial-dependent hypertension. Using data from a human clinical study, a mouse model with endothelial-specific deletion of argininosuccinate lyase (Asl), and in vitro studies in human aortic endothelial cells and induced pluripotent stem cell-derived endothelial cells from individuals with ASLD, we show that loss of ASL in endothelial cells leads to endothelial-dependent vascular dysfunction with reduced nitric oxide (NO) production, increased oxidative stress, and impaired angiogenesis. Our findings show that ASLD is a unique model for studying NO-dependent endothelial dysfunction in human hypertension.
Copyright © 2018 American Society of Human Genetics. Published by Elsevier Inc. All rights reserved.

Induced pluripotent stem cells (iPSCs) stand to revolutionize the way we study human development, model disease, and eventually, treat patients. However, these cell sources produce progeny that retain embryonic and/or fetal characteristics. The failure to mature to definitive, adult-type cells is a major barrier for iPSC-based disease modeling and drug discovery. To directly address these concerns, we have developed a chemically defined, serum and feeder-free-directed differentiation platform to generate hematopoietic stem-progenitor cells (HSPCs) and resultant adult-type progeny from iPSCs. This system allows for strict control of signaling pathways over time through growth factor and/or small molecule modulation. Through direct comparison with our previously described protocol for the production of primitive wave hematopoietic cells, we demonstrate that induced HSPCs are enhanced for erythroid and myeloid colony forming potential, and strikingly, resultant erythroid-lineage cells display enhanced expression of adult β globin indicating definitive pathway patterning. Using this system, we demonstrate the stage-specific roles of two key signaling pathways, Notch and the aryl hydrocarbon receptor (AHR), in the derivation of definitive hematopoietic cells. We illustrate the stage-specific necessity of Notch signaling in the emergence of hematopoietic progenitors and downstream definitive, adult-type erythroblasts. We also show that genetic or small molecule inhibition of the AHR results in the increased production of CD34+ CD45+ HSPCs while conversely, activation of the same receptor results in a block of hematopoietic cell emergence. Results presented here should have broad implications for hematopoietic stem cell transplantation and future clinical translation of iPSC-derived blood cells. Stem Cells 2018;36:1004-1019.
© 2018 The Authors STEM CELLS published by Wiley Periodicals, Inc. on behalf of AlphaMed Press.