Imaging the Dynamic Interaction Between Sprouting Microvessels and the Extracellular Matrix

Abstract

Thorough understanding of growth and evolution of tissue vasculature is fundamental to many fields of medicine including cancer therapy, wound healing, and tissue engineering. Angiogenesis, the growth of new vessels from existing ones, is dynamically influenced by a variety of environmental factors, including mechanical and biophysical factors, chemotactic factors, proteolysis, and interaction with stromal cells. Yet, dynamic interactions between neovessels and their environment are difficult to study with traditional fixed time imaging techniques. Advancements in imaging technologies permit time-series and volumetric imaging, affording the ability to visualize microvessel growth over 3D space and time. Time-lapse imaging has led to more informative investigations of angiogenesis. The environmental factors implicated in angiogenesis span a wide range of signals. Neovessels advance through stromal matrices by forming attachments and pulling and pushing on their microenvironment, reorganizing matrix fibers, and inducing large deformations of the surrounding stroma. Concurrently, neovessels secrete proteolytic enzymes to degrade their basement membrane, create space for new vessels to grow, and release matrix-bound cytokines. Growing neovessels also respond to a host of soluble and matrix-bound growth factors, and display preferential growth along a cytokine gradient. Lastly, stromal cells such as macrophages and mesenchymal stem cells (MSCs) interact directly with neovessels and their surrounding matrix to facilitate sprouting, vessel fusion, and tissue remodeling. This review highlights how time-lapse imaging techniques advanced our understanding of the interaction of blood vessels with their environment during sprouting angiogenesis. The technology provides means to characterize the evolution of microvessel behavior, providing new insights and holding great promise for further research on the process of angiogenesis.

Keywords: angiogenesis; extracellular matrix; neovessels; time-series imaging; vascular networks.

Figure 1

Schematic highlighting the interplay between the growing neovessel and the surrounding matrix structure. Obtained with permission from Hoying et al. (1996).

Figure 2

Microvascular networks observed at different levels of collagen density, and associated measurements about the network. Increasing the density of the ECM reduced neovascularization in both the experiments and computational simulations. Top row (A–C): Z-projection mosaic of 3D confocal image data showing vascularized collagen gels taken at Day 6 with different initial collagen concentration. (D–F) Results of comparable computational simulations, presented as renderings of the line segment data. (G) The total vascular length decreased as matrix density increased. Experimental measurements presented in black and computational predictions presented in gray. (H) Vessel interconnectivity, measuring percentage of microvessels that are connected into the largest continuous vascular network, decreased as a function of matrix density. (I) Branching point, defined as a node that connected to three or more vessel segments, was created by either a new vessel sprout (branching) or two separate vessels fusing into one (anastomosis). The number of branch points was normalized by the total vascular length to isolate the tendency of microvessels to branch as they grow. Branching per unit length decreased as matrix density increased. (J) An end point was defined as a node that was associated with only one vessel segment and represents the terminal end of a vessel. Normalizing the number of end points by the total vascular length revealed that the number of free ends per unit length increased with matrix density. There was a significant effect of matrix density on total vascular length, network connectivity, branch points, and free ends per unit length for both experimental and computational results (one-way ANOVA, p < 0.05 in all four cases). Modified with permission from Edgar et al. (2014).

Figure 3

Phase contrast light micrographs of rat microvessel fragments embedded in type I collagen matrix. This experimental model is used to represent angiogenesis in vitro. (Left) Isolated microvessel fragment at Day 0 with a visible lumen, indicated by the arrow. (Right) Angiogenic microvessel fragments at Day 6 of growth. The thicker initial fragments are indicated by the arrows. The thinner protrusions extending from the initial fragments are neovessels formed through angiogenesis. Obtained with permission from Edgar et al. (2014).

Figure 4

Experimental images and schematics of collagen fibril reorientation by the tips of growing neovessels. (A,B) Collagen fibrils (white) and endogenous endothelial cell fluorescence were visualized using SHG/two-photon microscopy. Scale bar = 20 μm. Yellow arrows point to new sprouts arising from the parent microvessel (green). (C,D) Schematics of collagen fibril orientation by neovessel tips, and potential mechanism of contact guidance. The “fan” of aligned matrix fibrils (red lines) in front of each growing neovessel would act to “track” the neovessels toward each other when overlapping. (D) Consequences to neovessel location due to differences in the size of the fibril-alignment zone. A small fibril-alignment zone would not readily overlap, while a larger zone (due to a less stiff matrix) would make overlapping of these tracks between neighboring neovessels more likely. Modified with permission from Edgar et al. (2014).

Figure 5

A sequence of images from time-lapse video of neovessel sprouting, growth, and inosculation within a collagen gel stroma. Microvessels (red) were imaged via confocal microscopy and collagen fibril structure (green) was visualized using SHG imaging. Over the course of ~4.5 days, a neovessel sprout (white arrow) forms, grows, and changes direction to eventually inosculate (wide arrow) with a second neovessel (open arrow) that appears from out of the field of view. Brackets indicate areas of collagen condensation occurring at neovessel walls. The asterisk marks a neovessel sprout that forms and then regresses approximately 3 days later. Obtained with permission from Edgar et al. (2014).

Figure 6

(A) Chemokinesis is increase in cellular movement proportional to cytokine concentration without oriented trajectories. (B) Chemotaxis is an increase in orientation of a cell’s trajectory in the presence of a cytokine gradient. (C) Boyden (transwell) chambers. The bottom chamber containing the chemoattractant and the top chamber containing the cells of interest are separated by a permeable membrane which can be removed and fixed to analyze chemotaxis. (D) Top view of a Dunn chamber. (E) Profile of Dunn chamber. The outer well containing chemoattractant and the inner well containing controlled media are separated by a central post, allowing a gradient to form between the two. Cells are seeded on glass slides flush with the innermost and outermost rings. (F) Simplified schematic of typical microfluidic chemotaxis chamber. A central source well (center left) is flanked by sink channels which form a gradient of chemoattractant from the centerline. Cells are seeded downstream and migrate to the centerline during chemotaxis. (G) Alternative schematic in which the top inlet contains the chemoattractant, the middle contains cells and media, and the bottom is a sink.

Figure 7

Role of stromal cells in angiogenesis. Macrophages and MSCs secrete MMPs, which degrade the matrix to allow sprouts to form and grow, and releases matrix bound growth factors. (A) Macrophages and MSCs secrete growth factors to stimulate sprouting and guide growing neovessels, and MSCs secrete factors to attract macrophages and modulate their phenotype. (B) Macrophages can facilitate neovessel fusion by bridging two tip cells. (C) Macrophages and MSCs can also act as pericytes to stabilize newly developed blood vessels. (D) MSCs also aid in lumen formation.

Figure 8

Time-lapse bioluminescence imaging of rats with implanted HUVECs or HUVECs + HMSCs. After an initial drop, the number of luciferin-activated cells increases over time to a greater extent in the HUVEC + HMSC group compared to the HUVEC only group. This suggests increased vascular network formation in the implant with HMSCs, compared to HUVECs alone. This figure was reproduced with permission from Elsevier (Sanz et al., 2008).

Figure 9

Mechanical and biophysical signals of a microvessel as it interacts with its microenvironment during growth. Microvessels are shown in red, new microvessel growth is shown in brighter red on the right column, and extracellular matrix fibers are shown in black. The first column of each section shows an arbitrary initial point in time when a vessel is first observed. The vessel remains the same across the rows but ECM conditions are altered. The second column represents a later point in time of the same vessel and its ECM microenvironment. (A) A microvessel forms cell-ECM adhesions, exerts pulling forces, and deforms the ECM during growth. (B) Higher ECM density, along with increased stiffness, causes microvessels to induce decreased deformation and decreased growth. (C) Stiffer ECM independent of fibril density leads to increased branching. (D) Pre-existing ECM fiber alignment induces directed neovessel growth.