Image-based crosstalk analysis of cell-cell interactions during sprouting angiogenesis using blood-vessel-on-a-chip.

BACKGROUND: Sprouting angiogenesis is an important mechanism for morphogenetic phenomena, including organ development, wound healing, and tissue regeneration. In regenerative medicine, therapeutic angiogenesis is a clinical solution for recovery from ischemic diseases. Mesenchymal stem cells (MSCs) have been clinically used given their pro-angiogenic effects. MSCs are reported to promote angiogenesis by differentiating into pericytes or other vascular cells or through cell-cell communication using multiple protein-protein interactions. However, how MSCs physically contact and move around ECs to keep the sprouting angiogenesis active remains unknown. METHODS: We proposed a novel framework of EC-MSC crosstalk analysis using human umbilical vein endothelial cells (HUVECs) and MSCs obtained from mice subcutaneous adipose tissue on a 3D in vitro model, microvessel-on-a-chip, which allows cell-to-tissue level study. The microvessels were fabricated and cultured for 10 days in a collagen matrix where MSCs were embedded. RESULTS: Immunofluorescence imaging using a confocal laser microscope showed that MSCs smoothed the surface of the microvessel and elongated the angiogenic sprouts by binding to the microvessel's specific microstructures. Additionally, three-dimensional modeling of HUVEC-MSC intersections revealed that MSCs were selectively located around protrusions or roots of angiogenic sprouts, whose surface curvature was excessively low or high, respectively. CONCLUSIONS: The combination of our microvessel-on-a-chip system for 3D co-culture and image-based crosstalk analysis demonstrated that MSCs are selectively localized to concave-convex surfaces on scaffold structures and that they are responsible for the activation and stabilization of capillary vessels.

A New Model for Specific Visualization of Skin Graft Neoangiogenesis Using Flt1-tdsRed BAC Transgenic Mice.

BACKGROUND: Neovascularization plays a critical role in skin graft survival. Up to date, the lack of specificity to solely track the newly sprouting blood vessels has remained a limiting factor in skin graft transplantation models. Therefore, the authors developed a new model by using Flt1-tdsRed BAC transgenic mice. Flt1 is a vascular endothelial growth factor receptor expressed by sprouting endothelial cells mediating neoangiogenesis. The authors determined whether this model reliably visualizes neovascularization by quantifying tdsRed fluorescence in the graft over 14 days. METHODS: Cross-transplantation of two full-thickness 1 × 1-cm dorsal skin grafts was performed between 6- to 8-week-old male Flt1 mice and KSN/Slc nude mice (n = 5). The percentage of graft area occupied by tdsRed fluorescence in the central and lateral areas of the graft on days 3, 5, 9, and 14 was determined using confocal-laser scanning microscopy. RESULTS: Flt1+ endothelial cells migrating from the transgenic wound bed into the nude graft were first visible in the reticular dermis of the graft center on day 3 (0.5 ± 0.1; p < 0.05). Peak neovascularization was observed on day 9 in the lateral and central parts, increasing by 2- to 4-fold (4.6 ± 0.8 and 4.2 ± 0.9; p < 0.001). Notably, some limited neoangiogenesis was displayed within the Flt grafts on nude mice, particularly in the center. No neovascularization was observed from the wound margins. CONCLUSION: The ability of the Flt1-tdsRed transgenic mouse model to efficiently identify the origin of the skin-graft vasculature and visualize graft neovascularization over time suggests its potential utility for developing techniques that promote graft neovascularization.