Stromal Cells Promote Neovascular Invasion Across Tissue Interfaces

Abstract

Vascular connectivity between adjacent vessel beds within and between tissue compartments is essential to any successful neovascularization process. To establish new connections, growing neovessels must locate other vascular elements during angiogenesis, often crossing matrix and other tissue-associated boundaries and interfaces. How growing neovessels traverse any tissue interface, whether part of the native tissue structure or secondary to a regenerative procedure (e.g., an implant), is not known. In this study, we developed an experimental model of angiogenesis wherein growing neovessels must interact with a 3D interstitial collagen matrix interface that separates two distinct tissue compartments. Using this model, we determined that matrix interfaces act as a barrier to neovessel growth, deflecting growing neovessels parallel to the interface. Computational modeling of the neovessel/matrix biomechanical interactions at the interface demonstrated that differences in collagen fibril density near and at the interface are the likely mechanism of deflection, while fibril alignment guides deflected neovessels along the interface. Interestingly, stromal cells facilitated neovessel interface crossing during angiogenesis via a vascular endothelial growth factor (VEGF)-A dependent process. However, ubiquitous addition of VEGF-A in the absence of stromal cells did not promote interface invasion. Therefore, our findings demonstrate that vascularization of a tissue via angiogenesis involves stromal cells providing positional cues to the growing neovasculature and provides insight into how a microvasculature is organized within a tissue.

Quantifying Vascular Density in Tissue Engineered Constructs Using Machine Learning

“…Whole, intact, microvessel fragments were isolated from adipose tissue, from either discarded human lipoaspirates or epididymal fat from male retired breeder Sprague Dawley rats (all animal procedures were approved by the Dartmouth College IACUC) and assembled into angiogenesis assays as previously described ( Nunes et al, 2013 ; Strobel et al, 2020 ). Rat vessels were cultured at 60 k/ml in DMEM (Gibco) containing 20% fetal bovine serum (FBS; Thermo Fisher), 1% penicillin-streptomycin (Fisher), and 1% amphotericin B (Fisher).…”

“…Assessments were made of four different treatments (Groups 1–4) promoting differing levels of angiogenesis. Experimental setups are reported in detail in Strobel et al (2020) . Briefly, for fluorescent 4× images, Group 1 contained microvessels with no additional stimuli, Group 2 was treated with vascular endothelial growth factor (VEGF), stromal cells were incorporated in Groups 3 and 4, and Group 4 was treated with a VEGF trap [R&D systems, described in Strobel et al (2020) ].…”

“…The variation in treatment groups was intended to demonstrate the utility of the BioSegment software. The data presented and discussed in this manuscript pertains to the accuracy of BioSegment measurements, not the scientific findings of these experiments, which are already published ( Strobel et al, 2020 ).…”

“…Thus, manual annotations were our primary validation method. In addition, we compared the BioSegment measurements to our in-house pixel-based protocol in which images are processed to improve contrast, thresholded, filtered to remove small cells and large clumps, and skeletonized to measure vessel lengths ( Strobel et al, 2020 ). The data reported are from either combined train and test datasets, or test-only datasets.…”

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“…Experimental setups are reported in detail in Strobel et al (2020). Briefly, for fluorescent 4× images, Group 1 contained microvessels with no additional stimuli, Group 2 was treated with vascular endothelial growth factor (VEGF), stromal cells were incorporated in Groups 3 and 4, and Group 4 was treated with a VEGF trap [R&D systems, described in Strobel et al (2020)]. For 10× images, Group 1 contained microvessels alone, Group 2 contained stromal cells in a separate region of collagen surrounding the microvessels, Group 3 contained stromal cells mixed in with the microvessels, and Group 4 contained stromal cells both around and within microvessel containing regions [described in detail in Strobel et al (2020)].…”