The primary findings from this study are that 1) during the initial stages of angiogenesis vascular islands undergo proliferation comparable to capillary sprouting and 2) during later stages of angiogenesis vascular islands are no longer present, in line with the hypothesis that these segments are able to incorporate into growing microvascular networks. In addition, vascular islands are associated with vascular pericytes, which have been attributed to play a role in capillary growth and stabilization [17
The mesentery was selected for this study because it allows for observation of intact microvascular networks (as compared to tissue cross-sections) with a resolution down to the single cell level. Use of the mesentery to investigate cellular dynamics during angiogenesis has served to identify endothelial cell phenotypic changes along capillary sprouts [18
], the relative positioning of pericytes along capillary sprouts [20
], and angiogenic pericyte phenotypes [10
]. The current study takes advantage of a robust model of angiogenesis stimulated by injections of compound 48/80, a mast cell degranulator [12
]. In the original description of this model, Norrby et al. demonstrated the ability of compound 48/80 to dramatically increase vascularized area, vascular density, and the number of vessels [12
]. The quantification of angiogenesis metrics in the current study also demonstrates these dramatic effects on microvascular network growth. Our characterization of vascular islands at different time points during growth further demonstrates the usefulness of this model and suggests a potential new mode of angiogenesis in an adult tissue involving endothelial cell proliferation and incorporation.
The origin of the endothelial cells along vascular islands is currently unknown emphasizing the need for future lineage studies. Potential sources could be attributed to the migration from existing vessels, a resident population of endothelial precursor cells, or vascular regression. Support for regression is provided by the observation of increased vascular islands during vascular pruning. In rat juvenile retinas hyperoxia was shown to increase the number of disconnected vascular segments. These segments were associated with endothelial cell apoptosis [5
]. In contrast, the vascular islands in rat mesenteric networks exhibited no evidence for positive TUNEL labeling (data not shown). Regardless of their origin, our results suggest that vascular islands can be triggered to enter a proliferative state.
Since proliferation along vascular islands was assessed with a single BRDU pulse, only the cells in S-phase at the time of tissue harvesting were captured. At day 3, the percentage of vascular islands with at least one proliferating cell was 33%. This value most likely underestimates the cellular proliferation associated with the vascular islands given that cells along the vascular islands are presumably in different stages of the cell cycle. Capillary sprouting is generally associated with endothelial cell proliferation. During peak capillary sprouting, the percentage of sprouts with a BRDU-positive cell was comparable to the percentage of vascular islands with a BRDU-positive cell. Thus, cells along vascular islands undergo proliferation similar to cells along capillary sprouts.
Previous dye injection experiments in the rat mesentery [21
] confirmed that capillary sprouts have lumens and that endothelial cell segments extend well past them. The structure of blood vascular islands is similar to these extending endothelial cell segments. Based on this and the dye injection data presented in this study, we hypothesize that vascular islands do not form lumens prior to connection to a nearby network.
A limitation of the current study is that individual vascular islands were not tracked over the time course of angiogenesis in the same tissue. This type of time lapse investigation is required to confirm the fate of vascular islands, to determine whether vascular islands increase their length, or whether the number of endothelial cells increases along a vascular island. In our current study, the average length of a vascular island was not different between the unstimulated and 3
days post stimulation scenario (data not shown). This lack of difference can be attributed to a high variability in both vascular island length and cell number. In spite of this heterogeneity, we do report that vascular islands form new branch points during angiogenesis. The increase in cellular extensions indicates that the vascular islands are dynamic and capable of changing their structure.
The increase in cellular extensions during angiogenesis combined with the increase in cell proliferation and the drastic reduction in number results provide strong support that vascular islands are able to incorporate into growing microvascular networks. However, how these vascular islands contribute to network growth remains unclear. Future experiments will be required to determine whether vascular islands actively direct the growth of nearby vessels or whether vascular islands represent a significant cell source for new vessels in an expanding network.
The ability of vascular islands to connect to a surrounding vascular network is supported by the endothelial cell dynamics observed during embryonic vasculogenesis. Progenitor cells aggregate and elongate into chord like formations subsequently producing vascular islands composed of endothelial cells [22
]. Over time these islands connect with each other and eventually to the surrounding vasculature, highlighting the ability of disconnected endothelial cell segments to connect to an existing network [22
]. In the adult, circulating endothelial progenitor cells (EPCs) have been suggested to incorporate into remodeling vessels [24
]. Additionally, microvessel fragments isolated from multiple adult tissues in vitro
are able to develop intact networks after implantation [27
]. These examples combined with our observations that the number of vascular islands decreases as vascular area and density increase support the hypothesis for incorporation of vascular islands into growing adult networks.
The involvement of vascular islands in angiogenesis is in line with a similar growth mechanism seen in lymphangiogenesis. Lymphatic vascular islands, identified as lymphatic endothelial cell segments disconnected from the surrounding network have been associated with proliferation, migration, and recruitment of cells during lymphangiogenesis [8
]. These lymphatic islands have the ability to eventually connect with the existing lymphatic network in which they are located [9
]. The overlapping mechanisms of lymphangiogenesis and angiogenesis include common cell phenotypes and responses to growth factors [9
]. The involvement of lymphatic islands in lymphangiogenesis implicates a role for blood vascular islands in angiogenesis.