An angiogenesis model for investigating multicellular interactions across intact microvascular networks

Abstract

Developing therapies aimed at manipulating microvascular remodeling requires a better understanding of angiogenesis and how angiogenesis relates to other network remodeling processes, such as lymphangiogenesis and neurogenesis. The objective of this study was to develop an angiogenesis model that enables probing of multicellular and multisystem interactions at the molecular level across an intact adult microvascular network. Adult male Wistar rat mesenteric windows were aseptically harvested and cultured in serum-free minimum essential media. Viability/cytotoxicity analysis revealed that cells remain alive for at least 7 days. Immunohistochemical labeling at 3 days for platelet endothelial cell adhesion molecule (PECAM), neuron-glial antigen 2 (NG2), lymphatic vessel endothelial hyaluronan receptor-1 (LYVE-1), and class III β-tubulin identified endothelial cells, pericytes, lymphatics, and nerves, respectively. Media supplemented with bFGF or VEGF induced an increase in endothelial cell sprouting off existing vessels. Endothelial cell sprouting in both growth factor groups was inhibited by targeting pericytes with NG2 functional blocking antibody. VEGF caused an increase in the number of lymphatic/blood endothelial cell connections compared with media alone or bFGF groups. Finally, the comparison of the same network before and after angiogenesis stimulated by the supplement of media with 20% serum identified the ability of disconnected endothelial segments to reconnect to nearby vessels. The results establish a novel in situ angiogenesis model for investigating the location of capillary sprouting within an intact network, the role of pericytes, lymphatic/blood endothelial cell interactions, and the fate of specific endothelial cell segments. The rat mesentery culture system offers a unique tool for understanding the complex dynamics associated with angiogenesis in an intact adult tissue.

Printing cancer cells into intact microvascular networks: a model for investigating cancer cell dynamics during angiogenesis

“…Blood versus lymphatic vessel identity was distinguishable by relative diameter, vessel morphology, and lectin and PECAM fluorescence labeling intensity. 10–12 Lymphatic identity based on these features is supported by our previous co-labeling of lymphatic vessels with lectin or PECAM and typical lymphatic markers: LYVE-1, 8,10–12 Prox1, 10–12 and podoplanin. 10–12 Arterioles, venules, and capillaries were identified by position in the network, vessel morphology, and labeling intensity as previously described.…”

“…8,9 Tissues were labeled with lectin for imaging at Day 0 and Day 2 or Day 5. Whole tissues were then mounted on glass slides, fixed in 100% methanol, and washed and permeabilized with 0.1% saponin in PBS.…”

Applications of computational models to better understand microvascular remodelling: a focus on biomechanical integration across scales

“…Example of an adult rat mesenteric microvascular network before and after angiogenesis. Time-lapse comparison of images from the same network was obtained using the rat mesentery culture model [1]. Asterisk (*) indicates regions of increased vessel density.…”

Hybrid Organic-Inorganic Scaffolding Biomaterials for Regenerative Therapies

“…It is well known that cell-cell interactions direct cellular activity towards these behaviors and contribute to determining the fate of uncommitted stem cells [10,11]. In addition, the material properties also directly affect cell function; in fact, these can be efficiently used to control cellular processes through three-dimensional chemical and physical…”