Topography of extracellular matrix mediates vascular morphogenesis and migration speeds in angiogenesis

Abstract

The extracellular matrix plays a critical role in orchestrating the events necessary for wound healing, muscle repair, morphogenesis, new blood vessel growth, and cancer invasion. In this study, we investigate the influence of extracellular matrix topography on the coordination of multi-cellular interactions in the context of angiogenesis. To do this, we validate our spatio-temporal mathematical model of angiogenesis against empirical data, and within this framework, we vary the density of the matrix fibers to simulate different tissue environments and to explore the possibility of manipulating the extracellular matrix to achieve pro- and anti-angiogenic effects. The model predicts specific ranges of matrix fiber densities that maximize sprout extension speed, induce branching, or interrupt normal angiogenesis, which are independently confirmed by experiment. We then explore matrix fiber alignment as a key factor contributing to peak sprout velocities and in mediating cell shape and orientation. We also quantify the effects of proteolytic matrix degradation by the tip cell on sprout velocity and demonstrate that degradation promotes sprout growth at high matrix densities, but has an inhibitory effect at lower densities. Our results are discussed in the context of ECM targeted pro- and anti-angiogenic therapies that can be tested empirically.

Figure 1. Model validation and geometry.

(A) The average extension speeds of our simulated sprouts agree with empirical measurements ,. Parameters were chosen to maximize sprout extension speeds. Reported speeds are an average of 10 independent simulations using the same parameter set. Error bars represent the standard error from the mean. The inset shows the two-dimensional 166 µm×106 µm geometry of the computational domain and simulated sprout development. Endothelial cells (red) migrate into the domain from a parent blood vessel (left boundary); a line source of VEGF diffuses from a tumor at the right boundary. The space between represents the stroma and is composed of extracellular matrix fibers (green) and interstitial fluid (blue). The sprout tip cell is identified with a T. (B) VEGF concentration profile (pg) showing higher concentrations of VEGF as the cells approach the tumor. (C) VEGF gradient profile (pg) is a better indicator of local VEGF heterogeneities. This image shows larger gradients in the proximity of the tip cell and along the leading edges of the new sprout. Snapshots at 7.8 hours.

Figure 2. Matrix density influences sprout velocity and branching.

(A) Dependence of average sprout extension speed on the density of the extracellular matrix. The model predicts that average extension speeds are maximal in the fiber fraction range . Above , extension speeds are significantly reduced and for and normal angiogenesis is interrupted suggesting that modulating matrix density may be an effective anti-angiogenesis therapy. (B) Quantification of morphological properties of the sprout showing sprout thicknesses within normal physiological ranges but dependent on matrix density and a distinct range of fiber density conductive to branching.

Figure 3. Plots showing the effect of the mechanical properties and heterogeneity of the ECM on sprout morphology and viability.

From top left to bottom right: (A) , interruption of normal angiogenesis and loss of sprout viability; (B) , high matrix heterogeneity induces branching (arrow points to new branch); (C) , anastomosis; (D) , more homogeneous matrix fiber network produces linear sprouts; (E) , higher matrix homogeneity causes loss of strong guidance cues resulting in wider and slower sprout formation; and (F) , complete inhibition of angiogenesis at high matrix density. Snapshots at 14 hours.

Figure 4. Evidence that mechanical cues, or contact guidance, from the ECM affects sprout extension.

(A) At , fiber network is not well connected and matrix fiber alignment does not have a strong effect on sprout extension speeds. At (B) and (C), rates of sprout extension are more rapid when matrix fibers are aligned parallel to VEGF gradients (0°) than when matrix fibers are aligned perpendicular to the gradient (90°).

Figure 5. Sprouts developing on patterned matrices reveal a strong correspondence between fiber alignment and cell shape and orientation.

Sprouts migrate toward higher concentrations of VEGF, however, cells elongate and are clearly oriented in the direction of the matrix cords. (A) Matrix of randomly distributed fibers augmented with horizontal cords 7.2 µm thick, (B) matrix cords 7.2 µm thick aligned horizontally, (C) horizontal cords 2.2 µm thick, (D) vertical cords 2.2 µm thick, and (E) crosshatched cords. Horizontal cords are aligned with to the VEGF gradient (0°); vertical cords are perpendicular to the gradient (90°); crosshatched cords form a ±45° angle with the VEGF gradient. These results demonstrate the important role of contact guidance and tissue structure in determining cell shape and orientation. Snapshots at 12.5 hours.

Figure 6. This plot shows that the effect of matrix degradation on average sprout extension speeds depends on the density of the ECM.

Solid lines represent average extension speeds without matrix degradation and the corresponding colored dashed lines show average speeds with tip cell matrix degradation. For , matrix degradation has anti-angiogenic effects. Above , degradation facilitates sprout progression.

Figure 7. Without degradation, angiogenesis is inhibited at (Figure 3F).

(A) shows that tip cell matrix degradation promotes sprout development at by carving out a path for migration, called a vascular guidance tunnel (B). (C) depicts sprout formation and branching with ECM degradation at , a density not typically conducive to branching, suggesting that high matrix heterogeneity (D) created by tip cell degradation may be a mechanism for branching (Video S3). (E) VEGF gradient profile (pg) shows strong gradient along leading edges of sprout. Snapshots at 14 hours.

Figure 8. Plot showing the effect of varying the chemotactic sensitivity parameter, , on average sprout extension speed at 14 hours.

Below , chemotactic cues are not strong enough relative to the energies associated with adhesion and growth to induce motility. Above , chemotactic incentives are so strong relative to adhesion and growth that the cells dissociate.