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. 2014 Sep;95:60-7.
doi: 10.1016/j.mvr.2014.06.009. Epub 2014 Jun 28.

Stretch-induced Intussuceptive and Sprouting Angiogenesis in the Chick Chorioallantoic Membrane

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Free PMC article

Stretch-induced Intussuceptive and Sprouting Angiogenesis in the Chick Chorioallantoic Membrane

Janeil Belle et al. Microvasc Res. .
Free PMC article

Abstract

Vascular systems grow and remodel in response to not only metabolic needs, but also mechanical influences as well. Here, we investigated the influence of tissue-level mechanical forces on the patterning and structure of the chick chorioallantoic membrane (CAM) microcirculation. A dipole stretch field was applied to the CAM using custom computer-controlled servomotors. The topography of the stretch field was mapped using finite element models. After 3days of stretch, Sholl analysis of the CAM demonstrated a 7-fold increase in conducting vessel intersections within the stretch field (p<0.01). The morphometric analysis of intravital microscopy and scanning electron microscopy (SEM) images demonstrated that the increase vessel density was a result of an increase in interbranch distance (p<0.01) and a decrease in bifurcation angles (p<0.01); there was no significant increase in conducting vessel number (p>0.05). In contrast, corrosion casting and SEM of the stretch field capillary meshwork demonstrated intense sprouting and intussusceptive angiogenesis. Both planar surface area (p<0.05) and pillar density (p<0.01) were significantly increased relative to control regions of the CAM. We conclude that a uniaxial stretch field stimulates the axial growth and realignment of conducting vessels as well as intussusceptive and sprouting angiogenesis within the gas exchange capillaries of the ex ovo CAM.

Keywords: Chorioallantoic membrane; Fluorescence microscopy; Microcirculation; Micromechanics; Scanning electron microscopy; Sprouting and intussusceptive angiogenesis; Stretch.

Figures

Figure 1
Figure 1
Schematic diagram of the chick chorioallantoic membrane (CAM) in ovo and after explantation (A). The change in CAM and embryo surface area (planar projection) in a representative ex ovo culture specimen is shown in B. The quasi-planar surface of a representative CAM is shown (planar projection) in C.
Figure 2
Figure 2
The stretch apparatus (A,B) used to apply the stretch field. B) Sutures attached to the EDD 10 chorioallantoic membrane (CAM) produced a uniaxial stretch field (black arrows); in control CAMs, the sutures were coupled to stationary motors. C) The result of the stretch field was a significant displacement of the suture attachment point in test CAMs (p<0.05)(stretch, closed circles; control, open circles). There was no significant difference in CAM surface area (D) or perimeter (E)(p>0.05, N=12).
Figure 3
Figure 3
Finite element models of the CAM stretch field. The Petri dish is assumed to reflect a static boundary; that is, these numerical simulations assumed that the shape of the CAM did not change. Internal forces (A,D) were assumed to be negligible. The simulations indicated modest displacement of the attachment points (B) with a linear stretch field reflected by the stress contour (C). The modification of the simulation to include a stiffer region of the CAM (D)—reflecting the presence of an embryo—had a moderate effect on both the displacement and stress contours (E,F). Of note, there was a prominent stress contour in the region of the “embryo” (F).
Figure 4
Figure 4
Sholl analysis of mosaic image reconstructions of the vascular pattern after 3 days (EDD 10 to 13) of stretch. Control CAM, with attachment points (circles) coupled to stationary servomotors, demonstrated little re-alignment of the CAM vessels (A,C). B) Test CAM stretched for 3 days demonstrated marked re-alignment of the conducting vessels within the stretch field (B,D). Variation in vessel location reflects normal CAM movements; variation in intravascular fluorescence reflects the time-dependent perfusion differences during multi-image acquisition. In panels C and D, closed circles are Sholl intersections in control CAM (N=7); open circles are Sholl intersections in CAM after 3 days of stretch (N=8). Radial sectors from -20° to +20° are indicated on the Sholl grid; bar =3mm.
Figure 5
Figure 5
Morphometry of the microcirculation in control (A) and stretched (C) CAM studied by fluorescence intravital microscopy of the stretch field. B) Interbranch distance was significantly greater in the stretch field than in controls (p<.05). D) Similarly, vessel branch angles were significantly decreased in the stretch field (p<.05)(N=15).
Figure 6
Figure 6
Corrosion casting and scanning electron microscopy (SEM) of the EDD 13 CAM microcirculation. Control CAM microcirculation demonstrated a delicate capillary meshwork observed from the conductive vessel (A) and capillary (B) surfaces. After the application of stretch for 3 days (EDD 10 to 13), the capillary meshwork demonstrated confluent growth of the network (C) with morphologic evidence of both sprouts and many intussusceptive pillars (D, ellipse).
Figure 7
Figure 7
Corrosion casting and scanning electron microscopy (SEM) of the EDD 13 CAM stretch field. Planar (2D) projections of the stretch field (A) were imaged; vessels were thresholded and surface area was calculated using morphometry software (MetaMorph). B) The fractional surface area was significantly greater in stretched compared to control CAM (p<.05). C) Higher resolution SEM demonstrated small pillars (arrows) often seen as doublets (ellipses). D) The number of pillars, expressed per 100μm2 was significantly higher in stretched CAMs compared to controls (N=6; p<.01).

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