To ensure survival of engineered implantable tissues thicker than approximately 2-3 mm, convection of nutrients and waste products to enhance the rate of transport will be required. Creating a network of vessels in vitro, before implantation (prevascularization), is one potential strategy to achieve this aim. In this study, we developed three-dimensional engineered vessel networks in vitro by coculture of endothelial cells (ECs) and fibroblasts in a fibrin gel for 7 days. Vessels formed by cord blood endothelial progenitor cell-derived ECs (EPC-ECs) in the presence of a high density of fibroblasts created an interconnected tubular network within 4 days, compared with 5-7 days in the presence of a low density of fibroblasts. Vessels derived from human umbilical vein ECs (HUVECs) in vitro showed similar kinetics. Implantation of the prevascularized tissues into immunecompromised mice, however, revealed a dramatic difference in the ability of EPC-ECs and HUVECs to form anastomoses with the host vasculature. Vascular beds derived from EPC-ECs were perfused within 1 day of implantation, whereas no HUVEC vessels were perfused at day 1. Further, while almost 90% of EPC-EC-derived vascular beds were perfused at day 3, only one-third of HUVEC-derived vascular beds were perfused. In both cases, a high density of fibroblasts accelerated anastomosis by 2-3 days. We conclude that both EPC-ECs and a high density of fibroblasts significantly accelerate the rate of functional anastomosis, and that prevascularizing an engineered tissue may be an effective strategy to enhance convective transport of nutrients in vivo.

Objective Angiogenesis requires tightly coordinated cross-talk between endothelial cells and stromal cells such as fibroblasts and smooth muscle cells. The specific molecular mechanisms moderating this process are still poorly understood. Method and Results Stromal cell-derived factors are essential for endothelial cell sprouting and lumen formation. We therefore compared the abilities of two primary fibroblast isolates and a primary smooth muscle cell isolate to promote in vitro angiogenesis and analyzed their secretomes using a combination of nanoLC-MS/MS, qPCR and ELISA. Each isolate exhibited a different level of angiogenic ability. Using quantitative MS, we then compared the secretomes of a fibroblast isolate exhibiting low angiogenic activity, a fibroblast isolate exhibiting high angiogenic activity and human umbilical vein endothelial cells. High angiogenic fibroblast supernatants exhibited an over-abundance of proteins associated with extracellular matrix constituents compared to low angiogenic fibroblasts or endothelial cells. Finally, siRNA technology and purified protein were used to confirm a role for stromal cell-derived hepatocyte growth factor and fibronectin in inducing endothelial cell sprouting. Conclusion Differences in stromal cell ability to induce angiogenesis are due to differences in the secreted proteomes of both extracellular matrix proteins and pro-angiogenic growth factors.

Objective It is well established that angiogenesis is a complex and coordinated multi-step process. However, there remains a lack of information about the genes that regulate individual stages of vessel formation. Here, we aimed to define the role of human interferon-induced transmembrane protein 1 (IFITM1) during blood vessel formation. Approach and Results We identified IFITM1 in a microarray screen for genes differentially regulated by endothelial cells (ECs) during an in vitro angiogenesis assay and found that IFITM1 expression was strongly induced as ECs sprouted and formed lumens. We showed by immunohistochemistry that human IFITM1 was expressed by stable blood vessels in multiple organs. siRNA-mediated knockdown of IFITM1 expression spared EC sprouting but completely disrupted lumen formation, both in vitro and in an in vivo xeno-transplant model. ECs lacking IFITM1 underwent early stages of lumenogenesis (i.e. intracellular vacuole formation) but failed to mature or expand lumens. Coimmunoprecipitation studies confirmed occludin as an IFITM1 binding partner in ECs and immunocytochemistry showed a lack of occludin at endothelial tight junctions in the absence of IFITM1. Finally, time-lapse video microscopy revealed that IFITM1 is required for the formation of stable cell-cell contacts during endothelial lumen formation. Conclusions IFITM1 is essential for the formation of functional blood vessels and stabilizes EC-EC interactions during endothelial lumen formation by regulating tight junction assembly.

The Interferon Inducible Transmembrane (IFITM) proteins were originally identified as part of a multi‐protein complex on the surface of lymphocytes that mediates homotypic adhesion and the transmission of antiproliferative signals. Recently, we have identified the IFITMs as regulators of endothelial cell (EC) sprouting in vitro, and angiogenesis in vivo. Angiogenesis is a multistep process whereby new blood vessels are formed from the preexisting vasculature in response to various angiogenic stimuli. During angiogenesis, EC shift from a quiescent phenotype, to migrating and proliferating cells as they sprout from parent vessels, and back to quiescent cells as the new vessels mature and stabilize. Both IFITM1 and IFITM2 are rapidly induced as EC sprout in vitro and form lumenized vessels, and knockdown of either disrupts both sprouting and lumen formation. Using a vascular bed xeno‐transplant model, we have also identified a role for the human IFITMs during in vivo angiogenesis. Immunohistochemical staining of multiple human tissues identified widespread IFITM expression on vessels. Our data suggest that IFITMs may regulate the transition of EC from a quiescent to an angiogenic state.