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.

Keywords: VEGF; neovessel invasion; stromal cells; tissue interface; vascular biology.

FIGURE 1

The core-in-field (CIF) tissue boundary model. (A) Schematic of the CIF model showing the preformed “core” sitting on a thin bed of gelled collagen surrounded by an additional “field” of gelled collagen. (B) Top-view of a phase microscopy image of a microvessel-free and cell-free CIF construct. (C) SEM image of a cross-section view of a CIF construct. (D) Higher-magnification of the interface and peri-interface region of a CIF construct. (E) Second harmonic generation image of the native collagen fibril structure comprising the interface and adjacent regions. (F) Fibril densities of the three regions in the CIF construct based on a validated method of measurement from SHG images. Bars are mean ± SD, N = 15, one-way ANOVA with Holm-Sidak post hoc analysis. *P < 0.05. In all cases, arrows indicate the interface between the core and field.

FIGURE 2

Simulations of neovessel growth in core-in-field models. (A) Top-view image of the interface of a CIF construct with microvessels growing along the tissue interface. White arrows point to the interface, black arrows point to microvessel being deflected along the interface. (B) Close-up of cut-view of cylindrical CIF geometry used in AngioFE. The core, interface, and field are colored in green, purple, and blue, respectively. Arrows indicate circumferential direction in the interface. (C) Simulations of vessel growth and behavior in CIF constructs with different interface densities and fibril organization. Visual results of the simulations for three different conditions of initial interface density (3 or 5 mg/mL) and fibril organization (anisotropic or isotropic) after 10 days of simulated culture. Cores are in light green, interfaces are in pink, fields are clear, and vessels are in red. (D) Log plot of predicted neovessel invasion across the interface for the three different simulated conditions, with an initial collagen concentration of either 3 or 4 mg/mL in the core. All groups are compared to the observed experimental value for microvessel-only constructs (white). Bars are mean ± SD, N = 4 for experiments, 10 for simulations. One-way ANOVA with Holm-Sidak post hoc analysis. ***P < 0.001 compared to experiments. There was an effect of interfacial matrix density on crossing regardless of core density. Further, simulated fibrils were more highly aligned than what was observed experimentally. (E) Validation of simulation mechanics determined by predicted final density after microvascular growth. Comparison of day 10 experimental (SHG) collagen densities after microvessel growth and contraction alongside predicted day 10 collagen densities from simulations. Simulations had an initial density in the core of 3 or 4 mg/mL. The initial interface and field densities were 5 and 3 mg/mL, respectively with isotropic fibrils for all simulations. The final densities predicted for each region by simulations are not different from those measured experimentally. One-way ANOVA performed on each region (core, interface, and field). Bars are mean ± SD. N = 15 (SHG) or N = 10 (simulation). P > 0.05.

FIGURE 3

Stromal vascular fraction cells promote neovessel crossing. (A) Without SVF cells, microvessels grow up to and then along the interface. (B) Inclusion of SVF cells results in neovessels crossing the interface to invade the field region. (C) SVF cells resulted in significant increases in neovessel crossing events, particularly when added to the field region. (D) Both cell number and spatial positioning affect crossing events. Bars are mean ± SEM, N = 4 (C) or N = 3 (D). One-way ANOVA with Newman–Keuls (C) or Tukey (D) post hoc analysis. *P < 0.05 compared to all other or specified groups. White arrows point to interface.

FIGURE 4

Stromal vascular fraction cells do not disrupt gross fibril structure of the interface. (A) Collagen fibril densities at and near the interface as measured by SHG imaging. Acellular CIF constructs are compared to microvessels cultured in CIF constructs (MV) and microvessels and SVF cells cultured for 10 days (MV + SVF). Bars are mean ± SD. N = 22, 15, and 10 for acellular, MV, and MV + SVF, respectively. Separate one-way ANOVA for densities in the core, interface, or field, with P > 0.05 in all cases. (B–D) SEM images of CIF constructs containing either microvessels (MV) or microvessels and SVF cells (MV + SVF). Arrows indicate the interface between the core and field. (D) is a higher magnification of the area in panel (C) highlighted by the dashed box. Arrows point to interface.

FIGURE 5

Stromal cell migration from the core to the field. (A,B) Confocal image stacks of CIF constructs formed with microvessels (red, rhodamine labeled Lectin stain) and SVF cells (green, GFP+) in the core after 10 days of culture. (C,D) Images of Hoechst stained samples without (C) or with (D) SVF incorporated into the core region. (E) Graph of Hoechst stained cells counted in the field region after 10 days of culture, with or without initial SVF incorporation in the core region. The circle, triangle, and box each represent a different experiment, with the line representing the mean of the experiments. Each experiment was statistically evaluated individually using a student’s t-test. P < 0.05 within the circle experiment and square experiment, but not the triangle experiment. In (A–D), white arrows point to microvessels, black arrows point to SVF. White dashed line indicates the interface.

FIGURE 6

Effect of VEGF-A on neovessel invasion. Effect of a VEGF trap on (A) interface crossings and (B) vessel density, compared SVF alone and a control IgG chimera protein. (C) Normalized neovessel invasion and (D) vessel density in CIF constructs containing microvessels alone or microvessels with recombinant VEGF-A₁₆₅ added to the media (+VEGF). One Way ANOVA with Newman–Keuls test (A,B) or Student’s t-test (C,D). Bars are mean ± SEM, N = 5. *P < 0.05 compared to all other groups.

FIGURE 7

Stromal cells in the core and field together reduce neovessel crossing. Crossing events when SVF is placed in both the core and field, compared to just the core or just the field. Squares, triangles, diamonds and +symbols represent means of 4 different experiments. Dashes represent the means of all experiments. A One-Way ANOVA with Newman–Keuls test was performed on groups within each individual experiment. P < 0.05 within the squares experiment for the no SVF compared to both field only and core only; within the triangle experiment for the field compared to all other groups; within the plus experiment for the field compared to no SVF. P < 0.05 for the diamond experiment.

FIGURE 8

Schematized hypothesis of how stromal cells guide growing neovessels across a tissue interface regardless of location relative to the growing neovessels.