Effect of mechanical boundary conditions on orientation of angiogenic microvessels

Abstract

Aim: Mechanical forces are important regulators of cell and tissue phenotype. We hypothesized that mechanical loading and boundary conditions would influence neovessel activity during angiogenesis.

Methods and results: Using an in vitro model of angiogenesis sprouting and a mechanical loading system, we evaluated the effects of boundary conditions and applied loading. The model consisted of rat microvessel fragments cultured in a 3D collagen gel, previously shown to recapitulate angiogenic sprouting observed in vivo. We examined changes in neovascular growth in response to four different mechanical conditions. Neovessel density, diameter, length and orientation were measured from volumetric confocal images of cultures exposed to no external load (free-floating shape control), intrinsic loads (fixed ends, no stretch), static external load (static stretch), or cyclic external load (cyclic stretch). Neovessels sprouted and grew by the third day of culture and continued to do so during the next 3 days of loading. The numbers of neovessels and branch points were significantly increased in the static stretch group when compared with the free-floating shape control group. In all mechanically loaded cultures, neovessel diameter and length distributions were heterogeneous, whereas they were homogeneous in shape control cultures. Neovessels were significantly more oriented along the direction of mechanical loading than those in the shape controls. Interestingly, collagen fibrils were organized parallel and adjacent to growing neovessels.

Conclusion: Externally applied boundary conditions regulate neovessel sprouting and elongation during angiogenesis, affecting both neovessel growth characteristics and network morphometry. Furthermore, neovessels align parallel to the direction of stress/strain or internally generated traction, and this may be because of collagen fibril alignment induced by the growing neovessels themselves.

Figure 1

(A) Mechanical conditioning system with raised platform mounted on motorized actuator. A stationary vertical stage is interfaced with the mobile anchor post by a beam. (B) Modified Labtek culture chamber with fixed anchor, mobile anchor supported by pins and removable Teflon mold. The Teflon mold was removed after collagen polymerization to yield a free floating tensile test specimen. An unanchored shape control (C) and a typical anchored construct (D). (E) Schematics show the four different test configurations in side view – (1) Shape Control, (2) No Stretch, (3) Static Stretch and (4) Cyclic Stretch.

Figure 2

(A) Six adjacent confocal image stacks were stitched together to obtain a mosaic of the center of the construct. (B) Final skeletonized output with largest continuous vessel highlighted

Figure 3

(A) 3D images of representative vascularized constructs: SC, Shape Control; NS, No Stretch; SS, Static Stretch; CS, Cyclic Stretch. (B) Construct vascularity and vascular morphometry measurements, expressed as total number of vessels per construct, volume fraction of total, number of vascular branch points and end points per construct and per vessel. Values are normalized to values of shape control. (Abbreviations: Tot. – Total for entire construct, Vssl. – Vessel, per continuous vessel, Vol. - Volume)

Figure 4

(A) Schematic of construct orientation convention. The long axis of the construct was aligned with direction of stretch or anchorage. (B) Projection of the segment along the Z-axis on the XY-plane was used to calculate projected angle (φ, Projected-X). (C) Orientation along X-axis (θ, horizontal, direction of stretch) and (D) Z-axis (δ, vertical, transverse to stretch) were directly determined based on segment orientation in these planes.

Figure 5

Segment length and diameter distributions. (A) Segment length distribution. Inset – Median segment lengths over treatment groups. (B) Segment length distribution across incremental angle bins. Orientation along the Projected-X direction is shown. (C) Segment diameter distribution within constructs. Inset – Median segment diameters over treatment groups. (D) Segment diameter distribution across incremental angle bins. Orientation along the Projected-X direction is shown.

Figure 6

Reorganization of collagen matrix by microvessels. Upper and lower panels contain representative images of the collagen matrix of gels without microvessels and gels with growing microvessels, subjected to identical stretching regimes after 6 days in culture. The collagen matrix in gels containing microvessels is more condensed making the individual collagen fibrils more difficult to distinguish. To eliminate bias due to collagen condensation around microvessels, the images selected for the lower panel do not contain microvessels in those fields. Scale bar in first image = 20 microns. SC, shape control; NS, no stretch; SS, static stretch; CS, cyclic stretch.