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Article by François Chesnais & Roberta Lock

Engineering Long-lasting Blood Vessels

Endothelial cells, the most inner layer of blood vessels, play a pivotal role in organ development, function, and regeneration. Thus, the creation of functional organ-specific blood vessels is of paramount importance for the engineering of tissues in vitro. However, endothelial cells from patients quickly lose their phenotype (i.e. their distinct and defining characteristics) in culture and cannot recreate mature vascular networks over a long period of time. To overcome this issue, the work presented here describes the “reset” of human endothelial cells by transiently overexpressing an embryonic transcription factor (proteins that help turn specific genes “on” or “off”), allowing for the formation of stable and physiologically relevant blood vessels.

What did these researchers do?

To enhance the formation of stable blood vessels in vitro, the group decided to “reset” endothelial cells (ECs) by transiently expressing the transcription factor ‘ETS variant transcription factor 2’ (ETV2). The transduction of ETV2 significantly increased the vasculogenic potential of ECs (meaning their ability to form blood vessels), leading to the formation of vascular networks stable for a long period of time both in vitro and in vivo, where control human ECs quickly degenerated. Importantly, “reset” vascular ECs (called R-VECs) were able to form blood vessels in mice, connect and integrate with the recipient vessels, and get covered with perivascular cells to form a stable vascular network.

The researchers then studied the molecular pathways involved in this process and discovered that a transient ETV2 overexpression allowed for a reset of the chromatin landscape (the 3D organization of DNA) in ECs to an adaptable and vasculogenic state, reminiscent of their phenotype during development in the embryo. Finally, the authors describe the use of R-VECs for the formation of engineered vascularized tissues. The vessels created in hydrogels from these ECs not only form perfusable vessels, but also create a capillary network that can be remodeled and respond to external stimuli to recreate physiologically relevant models of vasculature (such as how vasculature changes in disease, like cancer-associated vasculature).


Why is this important?

The maintenance of stable vascular networks in engineered tissues has been a central challenge in tissue engineering for decades. The lack of functional vasculature has limited the size of engineered tissues due to the oxygen diffusion limit and hindered the development of relevant in vitro models. This work paves the way for the development of better models to study the role of blood vessels on organ development and regeneration. It also supports the creation of perfusable organ-on-chip platforms of relevant size for toxicology, metabolic and immunological studies.

How did the researchers do this?

To switch their phenotype into a vasculogenic state, the authors induced a transient ETV2 expression via a lentivirus on human ECs in 2D culture. Then, these “reset” cells are detached and cultured in a 3D hydrogel to induce the self-assembly of capillaries in vitro or injected subcutaneously into mice to study in vivo vessel formation. Their main aim was to study the long-term formation of blood vessel and compare the stabilization of these vessels in both in vivo and in vitro settings. Then, they focused on the genes involved in this vascular resetting by RNA-sequencing. By comparing cells treated with a control lentivirus to the cells with a transient ETV2 expression, they studied genes involved in the different functions of endothelial cells, including angiogenesis (remodeling and expanding of vasculature network), lumen formation (the structure enabling transport of air, blood, fluids, or food), vasculogenesis (the differentiation and growth of blood vessels), and matrix remodeling (changing of the surrounding environment to support the cells), and found that the ETV2 overexpression brought the cells back to a primitive state.

Finally, the authors used these cells to create in vitro models by integrating the R-VECs within a hydrogel. First, the integration of R-VECs with human pancreatic islets inside a microfluidic device allowed their vascularization and the perfusion of whole blood to recapitulate physiological functions (e.g. glucose metabolism and insulin secretion). Then, the authors combined the R-VECs with either normal colon organoids or colorectal cancer organoids to study the capacity of R-VECs to adapt their phenotype to their environment. By comparing the R-VECs vasculogenic potential, proliferation and morphology as well as their transcription (gene expression) profile, they showed a remarkable adaptability of these cells, responding to external stimuli and recapitulating normal or malignant environments.


Human Pancreatic Islets (red) integrated in network of Blood Vessels (green) 

What comes next?

Although this study demonstrates the possibility to form stable and adaptable vascular networks both in vitro and in vivo, further investigations are needed to evaluate the functionality of these ECs. The authors describe the use of lentivirus on mature human ECs to regenerate their vasculogenic potential, which could be difficult to directly translate for human in vivo regeneration on clinical use.  Further investigation of the adaptability of these R-VECs is also needed to evaluate their ability to form functional organ-specific ECs with angiocrine signaling permeability or gene expression similar to mature ECs. Similar work has also reported the use of ETV2 overexpression in iPSCs, which could be useful to recreate patient-specific models and comparing the phenotypes in these different cell types could give insights on the parameters needed for the formation of stable vascular networks. Future efforts in the field will have to evaluate the effect of such transient overexpression on different vascular niches involved in organ development, regeneration and disease progression.  

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