Spatial control of angiogenesis by engineered patterns of Notch ligands

Laura Tiemeijer

Research output: Types of ThesisDoctoral ThesisCollection of Articles


In tissue engineering and regenerative medicine, proper vascularization of engineered tissues is imperative, as diffusion of nutrients, oxygen and waste is limited in constructs larger than a few cells thick. Therefore, tissue vascularization has been extensively researched. However, most approaches rely on preset structural support for the cells provided by scaffolds and micro fluidic chips to instruct vascular organization, which limit the integration into engineered and native tissues. Self-assembly of vascular constructs by combinations of support material, cells and growth factors shows promise but is not capable of dictating structure and organization of the engineered vasculatures. Therefore, spatial control over vascularization is required for tissue engineered constructs to remain alive and functioning.
The growth of vasculature from existing vessels towards the oxygen gradient, is a process called angiogenesis. During angiogenesis, migrating tip cells emerge from the vascular endothelium and sprout towards the oxygen gradient. Proliferating stalk cells follow their path and form a vascular tube. The selection of tip and stalk cell phenotypes is regulated via several cell signaling pathways, among which the Notch signaling pathwayhas a predominant role.
The Notch signaling pathway is a highly conserved cell-cell signaling pathway involved in numerous cell fate decisions and the patterning of tissues. The Notch receptor Notch1 and ligands Dll4 and Jag1 are involved in angiogenesis. Here, Dll4/Notch1 signaling is responsible for tip and stalk cell selection and Jag1 modulates Dll4/Notch1 signaling, though the role of Jag1 in angiogenesis needs to be further elucidated. Notch signaling is involved in several (genetic) cardiovascular diseases as well as cancer. For therapeutic targeting of Notch, mostly soluble molecules have been used to inhibit signaling. However, systemic administration of these molecules gives rise to adverse effects and toxicity. Local activation of Notch signaling, especially to target angiogenesis has not been widely addressed.
In this thesis, we have developed an in vitro method where we used spatial patterns of parallel lines of Notch signaling ligands to locally modulate endothelial tip/stalk cell selection and thereby gained spatial control over endothelial sprouting. The technical details of the protocol, optimization, important considerations and alternative approaches are also provided. Using the developed technology we have found that line patterns of Dll4 induced controlled unidirectional and restricted sprouting on the lines, resulting in localization of endothelial sprouts between the lines (negative patterning).
Next, we used our method to investigate the role of Jag1 in endothelial patterning. In contrast to Dll4, we have found that Jag1 patterns did not exert control over endothelial sprouting location and direction. Gene expression profiles did not provide significant difference upon stimulation with Dll4 and Jag1 to explain the difference in control observed.Computational modeling of Notch signaling endothelial cells on patterns of ligand, revealed that underlying differences in signaling dynamics between Jag1 and Dll4 are likely the cause for the lack of control over endothelial sprouting exerted by Jag1.
To study the potential of ligand patterning further, alternative techniques for complex patterning of ligands were considered and investigated, though further optimization is needed for their application.
In summary, we have shown that the use of local presentation of the Notch ligand Dll4 results in spatial control over endothelial sprouting. Additionally, the potential of engineered patterns of ligands to control endothelial sprouting during angiogenesis is valuable for the design of biomaterials and engineering of organized vasculature for tissue engineering as well as for regenerative medicine. Furthermore, the method developed in this thesis allows for the screening of functional engineered ligands and could aid in revealing disease phenotypes by screening for mutations in ligands.
Original languageEnglish
  • Sahlgren, Cecilia, Supervisor
  • Bouten, Carlijn V.C., Supervisor, External person
Place of PublicationTurku - Eindhoven
Print ISBNs 978-952-12-4218-2
Electronic ISBNs 978-952-12-4219-9
Publication statusPublished - 2022
MoE publication typeG5 Doctoral dissertation (article)

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