Embedded macrophages induce intravascular coagulation in 3D blood vessel-on-chip.

Macrophages are innate immune cells that prevent infections and help in wound healing and vascular inflammation. While these cells are natural helper cells, they also contribute to chronic diseases, e.g., by infiltrating the endothelial layer in early atherosclerosis and by promoting vascular inflammation. There is a crosstalk between inflammatory pathways and key players in thrombosis, such as platelets and endothelial cells - a phenomenon known as 'thromboinflammation'. The role of the embedded macrophages in thromboinflammation in the context of vascular disease is incompletely understood. Blood vessels-on-chips, which are microfluidic vascular cell culture models, have been used extensively to study aspects of vascular disease, like permeability, immune cell adhesion and thrombosis. Blood perfusion assays in blood vessel-on-chip models benefit from multiple unique aspects of the models, such as control of microvessel structure and well-defined flow patterns, as well as the ability to perform live imaging. However, due to their simplified nature, blood vessels-on-chip models have not yet been used to capture the complex cellular crosstalk that is important in thromboinflammation. Using induced pluripotent stem cell-derived endothelial cells and polarized THP-1 monocytes, we have developed and systematically set up a 3D blood vessel-on-chip with embedded (lipid-laden) macrophages, which is created using sequential cell seeding in viscous finger patterned collagen hydrogels. We have set up a human whole blood perfusion assay for these 3D blood vessels-on-chip. An increased deposition of fibrin in the blood vessel-on-chip models containing lipid-laden macrophages was observed. We anticipate the future use of this advanced vascular in vitro model in drug development for early atherosclerosis or aspects of other vascular diseases.

Extracellular vesicles produced by human-induced pluripotent stem cell-derived endothelial cells can prevent arterial stenosis in mice via autophagy regulation.

Intravascular transplantation of human-induced pluripotent stem cells (hiPSCs) demonstrated a significant therapeutic effect in the treatment of restenosis by the paracrine function of extracellular vesicles (EVs). However, the risk of tumorigenicity and poor cell survival limits its clinical applications. In this study, we for the first time applied a highly efficient and robust three-dimensional (3D) protocol for hiPSC differentiation into endothelial cells (ECs) with subsequent isolation of EVs from the derived hiPSC-EC (ECs differentiated from hiPSCs), and validated their therapeutic effect in intimal hyperplasia (IH) models. We found that intravenously (iv) injected EVs could accumulate on the carotid artery endothelium and significantly alleviate the intimal thickening induced by the carotid artery ligation. To elucidate the mechanism of this endothelial protection, we performed miRNA expression profiling and found out that among the most conserved endothelial miRNAs, miR-126 was the most abundant in hiPSC-EC-produced EVs (hiPSC-EC-EV). MiR-126 depletion from hiPSC-EC-EV can hinder its protective effect on human umbilical vein endothelial cells (HUVECs) in an inflammatory process. A variety of functional in vitro studies revealed that miR-126 was able to prevent endothelial apoptosis after inflammatory stimulation, as well as promote EC migration and tube formation through autophagy upregulation. The latter was supported by in vivo studies demonstrating that treatment with hiPSC-EC-EV can upregulate autophagy in mouse carotid artery ECs, thereby preventing IH and modulating vascular homeostasis via remodeling of the vascular intima. Our findings suggest a regulatory mechanism for the therapeutic effect on arterial restenosis by autophagy regulation, and provide a potential strategy for clinical treatment of the disease.

Exosomes Derived from Human Induced Pluripotent Stem Cells-Endothelia Cells Promotes Postnatal Angiogenesis in Mice Bearing Ischemic Limbs.

Induced pluripotent stem cell (iPSC) derived endothelial cells (ECs) is a novel therapeutic option for ischemic diseases. Although the detailed mechanism of this novel therapy remains unknown, emerging evidence has demonstrated that exosomes derived from hiPSC-ECs play a critical role in this approach. In this study, we first isolated and characterized the exosomes from iPSCs-ECs (hiPSC-EC-Exo) and determined the functional roles of hiPSC-EC-Exo in neovascularization and the underlying mechanism. Further, we evaluated the effect of exosomes derived from hiPS-ECs on promoting angiogenesis in a mouse model bearing ischemic limbs. Our results showed that miR-199b-5p, an miRNA highly associated with angiogenesis, is significantly upregulated during the differentiation of hiPSC-ECs. Mechanically, our studies found that hiPSC-ECs expressing miR-199b-5p significantly promote cell migration, proliferation and tube formation through Jagged-1-dependent upregulation of VEGFR2 in HUVECs. Similarly, coculture of hiPSC-ECs-Exo with HUVECs also resulted in a significant improvement in HUVEC migration, proliferation, and tube formation, suggesting that exosome-mediated cell-cell communication in a paracrine manner may serve as a fundamental mechanism for iPSC-EC-based treatment. Consequently, we found that the transfer of hiPSC-ECs enriched with miR-199b-5p significantly enhanced micro-vessel density and blood perfusion in ischemic limbs in vivo. Taken together, our studies were the first to demonstrate that transfer of hiPSC-ECs-Exo is a promising approach to treat ischemic injury via the mechanism of promoting neovascularization.