(316g) Engineering Functional Vascularization By Synthetic Regulation of Paracrine Signaling

The ever-increasing demand for tissue or organ transplants, coupled with a limited supply of suitable donors, motivates the development of engineered tissue replacements for transplantation into patients. However, the translational utility of engineered tissues is currently limited by the inability to create functional vasculature that can support the perfusion, survival, and function of clinically-relevant sizes of highly-metabolic tissues during in vitro culture or post-transplantation. While numerous strategies have delivered angiogenic growth factors or genes alongside engineered tissues during transplantation, these approaches have largely failed to establish perfused, long-lasting vasculature.

During development, the human body utilizes a process called vascular morphogenesis to create vasculature. Vascular morphogenesis is a multi-stage process, during which distinct phases of paracrine signaling between endothelial and stromal cells direct the assembly of endothelial cells into premature cords that eventually acquire lumen and organize into a perfused vascular network. Although paracrine factors that are characteristic of each stage of vascular morphogenesis have been identified, it is unknown how the magnitude and timing of signaling within each stage dictates vascular architecture and functionality.

In this work, we combined synthetic biology and tissue engineering tools to investigate how the magnitude and timing of paracrine signaling during vascular morphogenesis impacts the formation of functional vasculature. We engineered endothelial and stromal cell populations to express characteristic paracrine factors (e.g. VEGF, bFGF, etc.) from each stage of vascular morphogenesis. Each paracrine factor is installed under the control of an orthogonal synthetic zinc finger transcription regulator (synZiFTR), such that the expression of each factor is controllably induced by a different small molecule (e.g. grazoprevir, doxycycline, etc.). Through small molecule induction, we demonstrated independent and tunable control over the kinetics, concentration, and identity of paracrine factor production. We encapsulated these engineered cells in a microfluidic model of vascular morphogenesis, in which vascular architecture and functionality were evaluated by immunofluorescent staining and perfusion with fluorescent dextran respectively. Using different programs of paracrine signaling by varying the concentration, timing, and duration of each small molecule, we found that vascular density can be controlled by varying the magnitude of paracrine signaling in the early stages of vascular morphogenesis, while induction of late-stage signaling drives morphogenetic changes required for perfusion of the vascular network. Importantly, we identify a critical window of time for inducing late-stage signaling in order to maximize perfusion and vascular network formation.

Overall, these results provide insight into how paracrine signaling is regulated during vascular morphogenesis to give rise to functional vascular networks with varied architecture, and point to an approach to usurp these controls to control the vascularization of tissues. More broadly, the toolbox developed in this work for controlling paracrine signaling offers a new generalizable approach to investigate and uncover a wide variety of tissue morphogenetic programs that could be used to direct the assembly of artificial tissues.