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Objective: Emerging areas of vascular biology focus on lymphatic/blood vessel mispatterning and the regulation of endothelial cell identity. However, a fundamental question remains unanswered: Can lymphatic vessels become blood vessels in adult tissues? Leveraging a novel tissue culture model, the objective of this study was to track lymphatic endothelial cell fate over the time course of adult microvascular network remodeling. Methods: Cultured adult Wistar rat mesenteric tissues were labeled with BSI‐lectin and time‐lapse images were captured over five days of serum‐stimulated remodeling. Additionally, rat mesenteric tissues on day 0 and day 3 and 5 post‐culture were labeled for PECAM + LYVE‐1 or PECAM + podoplanin. Results: Cultured networks were characterized by increases in blood capillary sprouting, lymphatic sprouting, and the number of lymphatic/blood vessel connections. Comparison of images from the same network regions identified incorporation of lymphatic vessels into blood vessels. Mosaic lymphatic/blood vessels contained lymphatic marker positive and negative endothelial cells. Conclusions: Our results reveal the ability for lymphatic vessels to transition into blood vessels in adult microvascular networks and discover a new paradigm for investigating lymphatic/blood endothelial cell dynamics during microvascular remodeling. [ABSTRACT FROM AUTHOR]

A big problem associated with aging is thought to be impaired microvascular growth or angiogenesis. However, to link the evidence for impaired angiogenesis to microvascular dysfunction in aged tissues, we must compare adult vs. aged microvascular networks in unstimulated scenarios. The objective of this study was to test the hypothesis that aged microvascular networks are characterized by both fewer vessels and the impaired ability to undergo angiogenesis. Mesentery tissues from adult (9-mo) and aged (24-mo) male Fischer 344 rats were harvested and immunolabeled for platelet/endothelial cell adhesion molecule (an endothelial cell marker) according to two scenarios: unstimulated and stimulated. For unstimulated groups, tissues harvested from adult and aged rats were compared. For stimulated groups, tissues were harvested 3 or 10 days after compound 48/80-induced mast cell degranulation stimulation. Unstimulated aged microvascular networks displayed larger mean vascular area per tissue area compared with the unstimulated adult networks. The lack of a decrease in vessel density was supported at the gene expression level with RNA-Seq analysis and with comparison of vessel densities in soleus muscle. Following stimulation, capillary sprouting and vessel density were impaired in aged networks at 3 and 10 days, respectively. Our results suggest that aging associated with impaired angiogenesis mechanisms might not influence normal microvascular function, since unstimulated aged microvascular networks can display a "normal adult-like" vessel density and architecture., New & Noteworthy: Using a multidimensional approach, we present evidence supporting that aged microvascular networks display vessel density and patterning similar to adult networks despite also being characterized by a decreased capacity to undergo angiogenesis. Thus, vessel loss is not necessarily a characteristic of aging., (Copyright © 2017 the American Physiological Society.)

Angiogenesis, defined as the growth of new blood vessels from existing ones, plays a key role in development, growth, and tissue repair. Its necessary role in tumor growth and metastasis has led to the creation of a new category of anti-angiogenic cancer therapies. Preclinical development and evaluation of potential drug candidates require models that mimic real microvascular networks. Here, we describe the rat mesentery culture model as a simple ex vivo assay that offers time-lapse imaging of intact microvascular network remodeling and demonstrate its application for anti-angiogenic drug testing.

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.