Implanted microvessels progress through distinct neovascularization phenotypes

Abstract

We have previously demonstrated that implanted microvessels form a new microcirculation with minimal host-derived vessel investment. Our objective was to define the vascular phenotypes present during neovascularization in these implants and identify post-angiogenesis events. Morphological, functional and transcriptional assessments identified three distinct vascular phenotypes in the implants: sprouting angiogenesis, neovascular remodeling, and network maturation. A sprouting angiogenic phenotype appeared first, characterized by high proliferation and low mural cell coverage. This was followed by a neovascular remodeling phenotype characterized by a perfused, poorly organized neovascular network, reduced proliferation, and re-associated mural cells. The last phenotype included a vascular network organized into a stereotypical tree structure containing vessels with normal perivascular cell associations. In addition, proliferation was low and was restricted to the walls of larger microvessels. The transition from angiogenesis to neovascular remodeling coincided with the appearance of blood flow in the implant neovasculature. Analysis of vascular-specific and global gene expression indicates that the intermediate, neovascular remodeling phenotype is transcriptionally distinct from the other two phenotypes. Therefore, this vascular phenotype likely is not simply a transitional phenotype but a distinct vascular phenotype involving unique cellular and vascular processes. Furthermore, this neovascular remodeling phase may be a normal aspect of the general neovascularization process. Given that this phenotype is arguably dysfunctional, many of the microvasculatures present within compromised or diseased tissues may not represent a failure to progress appropriately through a normally occurring neovascularization phenotype.

Figure 1

Neovessel and network morphology in implanted microvascular constructs. A-D) Epifluorescence images of GFP-positive vessels in explanted constructs that were implanted for 7 (A), 14 (B) or 21 (C) days. Arrows in the d7 panel point to parent microvessel fragments that have given rise to numerous neovessel sprouts. Panel D is a light micrograph of a construct implanted for 28 days and filled, via the host circulation, with dialyzed ink followed by clearance with glycerol. E) Vessel segment quantification in implants. * ρ<0.05 as compared to day 7.

Figure 2

Cell proliferation in implanted constructs. Analysis of proliferation (A-C) and apoptosis (D-F) on histology sections of explanted constructs implanted for 7 (d7) or 28 (d28) days. (A and B) Sections immunostained for BrdU to identify proliferating cells (arrows). (C) The percentage of all cell nuclei positive for BrdU at each time point. * ρ<0.05 as compared to day 7 implants. (D and E) Sections stained by TUNEL show apoptotic cell nucleus in green (arrows) and nuclei staining by DAPI in blue. (F) The percentage of cell nuclei that were TUNEL positive at each time point. * ρ<0.05 as compared to day 14 implants.

Figure 3

Representative confocal images of microvessels in constructs en bloc immunostained for smooth muscle alpha actin (red) to identify perivascular cell morphology (A) in explants 7, 14, and 28 days after implantation. Endothelial cells were identified either by endogenous GFP fluorescence or staining with the lectin GS-1 conjugated to Alexa 488. A = arteriole; V = venule; arrow in day 7 panel indicates parent microvessel fragment. Characterization of network architecture in constructs at 7, 14, 21 and 28 days post implantation (B). Vessel diameter distribution in implants throughout network development. Measurements and statistical analysis were performed as described in the Methods section. Closed icons represent a significant difference in vessel diameter distribution (ρ<0.05) as compared to its respective open icon.

Figure 4

Perfusion of microvessels within implanted constructs. A-C) Merged representative confocal fluorescence images of implanted constructs explanted following rhodamine dextran injection into the host circulation showing the presence of the blood tracer dextran (red) inside GFP-positive construct vessels (green). D) Quantification of vessel perfusion. Vessels were considered perfused if positive for rhodamine-dextran. * ρ<0.05.

Figure 5

Blood perfusion and vessel morphology in constructs implanted for 14 or 28 days. A - D) Representative still images from real-time epifluorescence video-microscopy of exposed, day 14 (A, B) or day 28 (C, D) implanted constructs following an injection of a rhodamine-dextran blood tracer into the host circulation. Open arrow in panel B show vessels within the developing network lacking dextran fluorescence and, therefore, considered not perfused by blood. Panels A and B are also examples of perfused microvessels with atypical morphologies while panels C and D are examples of mature network morphologies. Closed arrows show the direction of blood flow as seen in the videos. The asterisk in A is adjacent to a vessel connection that spontaneously constricted during visualization. Micron bar shown is the same for all panels.

Figure 6

Gene expression patterns in the implanted microvascular constructs. Each data point in the plots is the mean and standard deviation of the relative mean-centered and unit-normalized expression values (scaled from 1.0 to −1.0) for each time point (d7, d14, d21, and d28) for the total number of the genes (lower right corner) in the pattern. In all cases, expression was normalized to the day 0 measurements, which was set to “0”. Letters in the upper right corner refer to the pattern ID and are addressed in the text. Select genes belonging to each pattern are shown to the right of the pattern. Gene transcripts measured by real-time PCR (see Supplemental Figure 2) are shown in italics.

Figure 7

Graphical results of the principal component analysis of all of the differentially expressed genes in the neovascularizing constructs. A) Plots of the relative impact of gene expression (Y-axis) occurring at each time point (X-axis) on the 3 primary principal components. The higher the Y-axis value for a given time point, the greater the contribution of that time point to the principal component. B) A 3-coordinate plot of the overall expression for each time point on the three main principal components (corresponding to the three different axes). Overall gene expression for the day 21 and 28 time points map together (blue circle) along the 3^rd principal component (PC 3) axis. While those for day 14 (red circle) and day 7 (arrow) map on the 1^st and 2^nd principal component (PC1 and PC2) axes, respectively. In this plot, the larger the data point, the closer it is to the reader.

Figure 8

Summary of the three phenotypes and two transitional activities in the neovascularizing microvascular construct.