Objective-We have previously demonstrated the ability to construct 3-dimensional microvascular beds in vitro via angiogenesis from isolated, intact, microvessel fragments that retain endothelial cells and perivascular cells. Our objective was to develop and characterize an experimental model of tissue vascularization, based on the implantation of this microvascular construct, which recapitulated angiogenesis, vessel differentiation, and network maturation. Methods and Results-On implantation in a severe combined-immunodeficient mouse model, vessels in the microvascular constructs rapidly inosculated with the recipient host circulation. Ink perfusion of implants via the left ventricle of the host demonstrated that vessel inosculation begins within the first day after implantation. Evaluation of explanted constructs over the course of 28 days revealed the presence of a mature functional microvascular bed. Using a probe specific for the original microvessel source, 91.7%Ϯ11% and 88.6%Ϯ19% of the vessels by day 5 and day 28 after implantation, respectively, were derived from the original microvessel isolate. Similar results were obtained when human-derived microvessels were used to build the microvascular construct. Key Words: vascularization Ⅲ microcirculation Ⅲ angiogenesis Ⅲ human Ⅲ vascular remodeling V ascularization is the process by which perfusion pathway length and vessel segment number are increased and organized into a functional vascular bed. In normal situations, this effective increase in vessel density delivers more blood to the tissue, facilitating tissue growth and/or increased tissue activity. 1,2 Consequently, vascularization is a primary component of tissue growth and repair, such as occurs during development, 3 after an upstream occlusive event leading to tissue ischemia, 4 or during proliferative events, as seen in tissue healing 5,6 and tumor growth. 7 Although we know of many factors and signals that initiate or terminate the vascularization process, little is known about the rules that govern vascularization as an integrated process that includes angiogenesis, 3 arteriogenesis, 8 vascular remodeling, 9 vessel adaptation, 10 and arterio-venous polarization. 11 We have previously shown that isolated intact microvessel fragments retain angiogenic potential and are capable of forming a simple microvascular bed when cultured in a 3-dimensional collagen I gel. 12 In this microvascular construct, the vessel fragments undergo stereotypical angiogenesis, forming neovessels that maintain patent lumen and perivascular cell associations. Furthermore, the vessel fragments within this culture system are responsive to proangiogenic conditions. 12,13 All of this occurs in the absence of blood flow and relatively few nonvascular cells. Conclusions-WithHere we report the development and characterization of an experimental model of tissue vascularization based on the implantation of this microvascular construct. Precultured or freshly formed microvascular constructs implanted subcutaneously inosculate with the...

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 vascularspecific 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.

Thrombin is a serine protease that promotes platelet aggregation, blood coagulation, and tissue repair. A peptide derived from a non-proteolytically active region of thrombin, TP508, also promotes tissue repair and increased vascularity, yet does not activate platelet and inflammatory cascades. TP508 binds to cells with high affinity and stimulates cells independent of the proteolytically active thrombin receptors (PARs) and thus is considered to activate a non-proteolytically active receptor (non-PAR) pathway. Using a model of angiogenic sprouting, we further defined the angiogenic potential of TP508 and investigated the role of non-proteolytic, thrombin-mediated pathways in angiogenesis. The assay involves measuring angiogenic sprouting from cultured, intact microvessel fragments. In this assay, TP508 stimulated angiogenic sprouting to an extent similar to or greater than the potent angiogenic factor, VEGF. However, TP508 had no significant effect on the number of sprouts that formed per vessel. In contrast to TP508, proteolytically active receptor agonists had no effect or inhibited angiogenic sprouting. The increased sprouting activity stimulated by TP508 was VEGF dependent but did not involve an increase in VEGF mRNA expression above baseline levels. These results suggest that TP508 acts early in angiogenesis and directly on microvascular cells to accelerate sprouting, but not to induce more sprouting, in a manner different than the intact thrombin molecule.