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In vitro models of angiogenesis are valuable tools for understanding the underlying mechanisms of pathological conditions and for the preclinical evaluation of therapies. Our laboratory developed the rat mesentery culture model as a new tool for investigating mechanistic cell–cell interactions at specific locations across intact blood and lymphatic microvascular networks ex vivo. The objective of this study was to report a method for evaluating the effect of aging on human stem cell differentiation into pericytes during angiogenesis in cultured microvascular networks. DiI labeled exogenous stem cells were seeded onto harvested adult Wistar rat mesenteric tissues and cultured in alpha-MEM + 1% serum for up to 5 days according to four experimental groups: (1) adult human adipose–derived stem cells (hASCs), (2) aged hASCs, (3) adult human bone marrow-derived stem cells (hBMSCs), and (4) aged hBMSCs. Angiogenesis per experimental group was supported by observation of increased vessel density and capillary sprouting. For each tissue per experimental group, a subset of cells was observed in typical pericyte location wrapped along blood vessels. Stem cell differentiation into pericytes was supported by the adoption of elongated pericyte morphology along endothelial cells and positive NG2 labeling. The percentage of cells in pericyte locations was not significantly different across the experimental groups, suggesting that aged mesenchymal stem cells are able to retain their differentiation capacity. Our results showcase an application of the rat mesentery culture model for aging research and the evaluation of stem cell fate within intact microvascular networks.

Microvascular network growth and remodeling are common denominators for most age-related pathologies. For multiple pathologies (myocardial infarction, stroke, hypertension), promoting microvascular growth, termed angiogenesis, would be beneficial. For others (cancer, retinopathies, rheumatoid arthritis), blocking angiogenesis would be desirable. Most therapeutic strategies, however, are motivated based on studies using adult animal models. This approach is problematic and does not account for the impaired angiogenesis or the inherent network structure changes that might result from age. Considering the common conception that angiogenesis is impaired with age, a need exists to identify the causes and mechanisms of angiogenesis in aged scenarios and for new tools to enable comparison of aged versus adult responses to therapy. The objective of this article is to introduce opportunities for advancing our understanding of angiogenesis in aging through the discovery of novel cell changes along aged microvascular networks and the development of novel ex vivo models.

A challenge in cancer research is the lack of physiologically responsive in vitro models that enable tracking of cancer cells in tissue-like environments. A model that enables real-time investigation of cancer cell migration, fate, and function during angiogenesis does not exist. Current models, such as 2D or 3D in vitro culturing, can contain multiple cell types, but they do not incorporate the complexity of intact microvascular networks. The objective of this study was to establish a tumor microvasculature model by demonstrating the feasibility of bioprinting cancer cells onto excised mouse tissue. Inkjet-printed DiI(+) breast cancer cells on mesometrium tissues from C57Bl/6 mice demonstrated cancer cells' motility and proliferation through time-lapse imaging. Colocalization of DAPI(+) nuclei confirmed that DiI(+) cancer cells remained intact postprinting. Printed DiI(+) 4T1 cells also remained viable after printing on Day 0 and after culture on Day 5. Time-lapse imaging over 5 days enabled tracking of cell migration and proliferation. The number of cells and cell area were significantly increased over time. After culture, cancer cell clusters were colocalized with angiogenic microvessels. The number of vascular islands, defined as disconnected endothelial cell segments, was increased for tissues with bioprinted cancer cells, which suggests that the early stages of angiogenesis were influenced by the presence of cancer cells. Bioprinting cathepsin L knockdown 4T1 cancer cells on wild-type tissues or nontarget 4T1 cells on NG2 knockout tissues served to validate the use of the model for probing tumor cell versus microenvironment changes. These results establish the potential for bioprinting cancer cells onto live mouse tissues to investigate cancer microvascular dynamics within a physiologically relevant microenvironment. IMPACT STATEMENT: To keep advancing the cancer biology field, tissue engineering has been focusing on developing in vitro tumor biomimetic models that more closely resemble the native microenvironment. We introduce a novel methodology of bioprinting exogenous cancer cells onto mouse tissue that contains multiple cells and systems within native physiology to investigate cancer cell migration and interactions with nearby microvascular networks. This study corroborates the manipulation of different exogenous cells and host microenvironments that impact cancer cell dynamics in a physiologically relevant tissue. Overall, it is a new approach for delineating the effects of the microenvironment on cancer cells and vice versa.

Restoration of form and function requires apposition of tissues in the form of flaps to reconstitute local perfusion. Successful reconstruction relies on flap survival and its integration with the recipient bed. The flap's precariously perfused hypoxic areas undergo adaptive microvascular changes both internally and in connection with the recipient bed. A cell-mediated, coordinated response to hypoxia drives these adaptive processes, restoring a tissue's normoxic homeostasis via de novo vasculogenesis, sprouting angiogenesis, and stabilizing arterialization. As cells exert prolonged and coordinated effects on site, their use as biological agents merit translational consideration of sourcing angio-competent cells and delivering them to territories enduring microcirculatory acclimatization. Angio-competent cells abound in adipose tissue: a reliable, accessible, and expendable source of adipose-derived cells (ADC). When subject to enzymatic digestion and centrifugation, adipose tissue separates its various ADC: A subset of buoyant oil-dense adipocytes (the tissue's parenchymal component) accumulates on a supra-natant layer, whereas the mesenchymal component remains in the infra-natant sediment, containing the tissue's stromal vascular fraction (SVF), where angio-component cells abound. The SVF can be further manipulated, selected, or culture expanded into more specific stromal subsets (herein defined as adipose stromal cells, ASC). While promising clinical applications for ADC await clinical proof and regulatory authorization, basic science investigation is needed to elucidate the specific ADC mechanisms that influence microvascular growth, remodeling, and function following flap surgery. The objective of this article is to share the clinical perspectives of reconstructive plastic surgeons regarding the use of ADC-based therapies to help with flap tissue integration, revascularization, and wound healing. Specifically, the focus will be on considering the potential for ADC as therapeutic agents and how their clinical application motivates basic science opportunities.

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.

Ageing is the main risk factor for the development of cardiovascular diseases. A central mechanism by which ageing promotes vascular pathologies is compromising endothelial health. The age-related attenuation of endothelium-dependent dilator responses (endothelial dysfunction) associated with impairment of angiogenic processes and the subsequent pathological remodelling of the microcirculation contribute to compromised tissue perfusion and exacerbate functional decline in older individuals. This Review focuses on cellular, molecular, and functional changes that occur in the endothelium during ageing. We explore the links between oxidative and nitrative stress and the conserved molecular pathways affecting endothelial dysfunction and impaired angiogenesis during ageing. We also speculate on how these pathological processes could be therapeutically targeted. An improved understanding of endothelial biology in older patients is crucial for all cardiologists because maintenance of a competently functioning endothelium is critical for adequate tissue perfusion and long-term cardiac health.

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.

A challenge for engineering models of angiogenesis is mimicking the physiological complexity of real microvascular networks. Utilizing an alternative top-down tissue culture approach, our laboratory developed the rat mesentery culture model as an ex vivo platform for investigating the multicellular dynamics involved during angiogenesis within an intact microvascular network. The objective of this study was to introduce physiologically relevant microvascular perfusion in cultured rat mesentery tissues and demonstrate its effect on angiogenesis. Adult male Wistar rat mesenteric tissues were harvested along with the main feeding artery and vein, and then transferred to a custom-designed biochamber for perfusion. The main feeding artery was cannulated with a 30G needle and secured in place with 7–0 suture. Single-pass perfusion was accomplished using a peristaltic pump in series with the biochamber placed inside an incubator set to standard culture conditions (37°C and 5% CO(2)). Flow passed through the vasculature and drained out of the venous side to be collected in a waste reservoir. Tissues were cultured for 48 h with perfusion in the biochamber (Perfused) in serum-supplemented media to stimulate angiogenesis. Control tissues were cultured in biochambers without perfusion (Static). Injection with FITC-albumin through the cannulated artery identified the lumens of vessels across the hierarchy of intact microvascular networks and confirmed successful perfusion. Labeling with BSI-lectin identified endothelial cells along microvascular networks and confirmed perfused tissues undergo angiogenesis after 48 h in culture, characterized by an increase in capillary sprouting. The presence of physiologic levels of capillary fluid velocities and associated shear stresses attenuated the angiogenic response compared to static controls. These results demonstrate the effect of perfusion on angiogenesis and establishes the novelty of the rat mesentery culture model as an experimental platform that incorporates perfusion with real microvascular networks in an ex vivo environment. IMPACT STATEMENT: Microvascular remodeling, or angiogenesis, plays a central role in multiple pathological conditions, including cancer, diabetes, and ischemia. Tissue-engineered in vitro models have emerged as tools to elucidate the mechanisms that drive the angiogenic process. However, a major challenge with model development is recapitulating the physiological complexity of real microvascular networks, including incorporation of the entire vascular tree and hemodynamics. This study establishes a bioreactor system that incorporates real microvascular networks with physiological flow as a novel ex vivo tissue culture model, thereby providing a platform to evaluate angiogenesis in a physiologically relevant environment.

Background Neurovascular patterning is an emerging area of microvascular research. While overlapping molecular signals highlight links between angiogenesis and neurogenesis, advancing our understanding is limited by a lack of in vitro models containing both systems. One potential model is the rat mesentery culture model, which our laboratory has recently introduced as an ex vivo tool to investigate cellular dynamics during angiogenesis in a microvascular network scenario. The objective of this study was to demonstrate the use of the rat mesentery culture model as an ex vivo platform for maintaining the spatiotemporal relationship between blood vessels and peripheral nerves during angiogenesis in adult microvascular networks. Methods Adult male Wistar rat mesenteric tissue windows were harvested, rinsed in sterile DPBS and medium and then cultured per group: 1) MEM alone and 2) NBM with NGF and 20 % FBS (nerve culture medium). On day 3 post culture tissues were labeled for endothelial (PECAM) and neural (class III β-tubulin, NG2, tyrosine hydroxylase, CGRP) markers. Results In MEM alone tissues nerve segment degeneration was supported by discontinuous nerve or absence of nerve marker labeling. Nerve presence at the arteriole level and capillary level was maintained for the nerve culture medium group compared to day 0, non-cultured control group (unstimulated). Comparison with existing methods and conclusions The results support the use of specific medium types to maintain nerve presence across cultured microvascular networks and implicates the rat mesentery culture model as a novel ex vivo tool for investigating neurovascular patterning in adult tissues.

A challenge in tissue engineering biomimetic models for studying angiogenesis is building the physiological complexity of real microvascular networks. Our laboratory recently introduced the rat mesentery culture model as an ex vivo experimental platform for investigating multicellular dynamics involved in angiogenesis within intact microvascular networks. The objective of this study was to compare endothelial cell phenotypes along capillary sprouts in cultured ex vivo rat mesentery microvascular networks to in vivo endothelial cell phenotypes. For Day 3 (Ex Vivo) tissues, adult rat mesentery tissues were cultured for three days in media supplemented with 10% serum. For Day 3 (In Vivo) tissues, adult rats were anesthetized and the mesentery was exteriorized for twenty minutes to induce angiogenesis. Microvascular networks from Day 3 (Ex Vivo) and Day 3 (In Vivo) groups were angiogenic, characterized by an increase in vessel density, capillary sprouting, and identification of similar BrdU-positive endothelial cell distributions along sprouts. Endothelial cells in both groups extended pseudopodia at the distal edge of capillary sprouts and displayed similar endothelial cell UNC5b, VEGFR-2, and CD36 labeling patterns. The results from this study support the physiological relevance of the rat mesentery culture model and highlight its novelty as a biomimetic tool for angiogenesis research.

Background: The development of models that incorporate intact microvascular networks enables the investigation of multicellular dynamics during angiogenesis. Our laboratory introduced the rat mesentery culture model as such a tool, which would be enhanced with mouse tissue. Since mouse mesentery is avascular, an alternative is mouse mesometrium, the connective tissue of uterine horns. The study’s objective was to demonstrate that mouse mesometrium contains microvascular networks that can be cultured to investigate multicellular dynamics during angiogenesis. Methods: Harvested mesometrium tissues from C57Bl/6 female mice were cultured in media with serum for up to 7 days. PECAM, NG2, αSMA, and LYVE-1 labeling identified endothelial cells, pericytes, smooth muscle cells, and lymphatic endothelial cells, respectively. Results: These cells comprised microvascular networks with arterioles, venules, and capillaries. Compared to day 0, capillary sprouts per vascular length were increased by 3 and 5 days in culture (day 0, 0.08 ± 0.01; day 3, 3.19 ± 0.78; day 5, 2.49 ± 0.05 sprouts/mm; p < 0.05). Time-lapse imaging of cultured tissues from FlkEGFP mice showcases the use of the model for lineage studies. The impact is supported by the identification of endothelial cell jumping from one sprout to another. Conclusion: These results introduce a novel culture model for investigating multicellular dynamics during angiogenesis in real-time ex vivo microvascular networks.

An emerging challenge in tissue engineering biomimetic models is recapitulating the physiological complexity associated with real tissues. Recently, our laboratory introduced the rat mesentery culture model as an ex vivo experimental platform for investigating the multi-cellular dynamics involved in angiogenesis within an intact microvascular network using time-lapse imaging. A critical question remains whether the vessels maintain their functionality. The objective of this study was to determine whether vascular smooth muscle cells in cultured microvascular networks maintain the ability to constrict. Adult rat mesenteric tissues were harvested and cultured for three days in either MEM or MEM plus 10% serum. On Day 0 and Day 3 live microvascular networks were visualized with FITC conjugated BSI-lectin labeling and arteriole diameters were compared before and five minutes after topical exposure to vasoconstrictors (50 mM KCl and 20 nM Endothelin-1). Arterioles displayed a vasoconstriction response to KCl and endothelin for each experimental group. However, the Day 3 serum cultured networks were angiogenic, characterized by increased vessel density, and displayed a decreased vasoconstriction response compared to Day 0 networks. The results support the physiological relevance of the rat mesentery culture model as a biomimetic tool for investigating microvascular growth and function ex vivo.

Angiogenesis, defined as the growth of new blood vessels from pre-existing vessels, involves endothelial cells, pericytes, smooth muscle cells, immune cells, and the coordination with lymphatic vessels and nerves. The multi-cell, multi-system interactions necessitate the investigation of angiogenesis in a physiologically relevant environment. Thus, while the use of in vitro cell-culture models have provided mechanistic insights, a common critique is that they do not recapitulate the complexity associated with a microvascular network. The objective of this protocol is to demonstrate the ability to make time-lapse comparisons of intact microvascular networks before and after angiogenesis stimulation in cultured rat mesentery tissues. Cultured tissues contain microvascular networks that maintain their hierarchy. Immunohistochemical labeling confirms the presence of endothelial cells, smooth muscle cells, pericytes, blood vessels and lymphatic vessels. In addition, labeling tissues with BSI-lectin enables time-lapse comparison of local network regions before and after serum or growth factor stimulation characterized by increased capillary sprouting and vessel density. In comparison to common cell culture models, this method provides a tool for endothelial cell lineage studies and tissue specific angiogenic drug evaluation in physiologically relevant microvascular networks.

Objective Lymphatic and blood microvascular systems are critical for tissue function. Insights into the coordination of both systems can be gained by investigating the relationships between lymphangiogenesis and angiogenesis. Recently, our laboratory established the rat mesentery culture model as a novel tool to investigate multicellular interactions during angiogenesis in an intact microvascular network scenario. The objective of this study was to determine whether the rat mesentery culture model can be used to study lymphangiogenesis. Methods Mesenteric tissue windows were harvested from adult male Wistar rats and cultured for three or five days in either serum-free MEM or MEM supplemented with VEGF-C. Tissues were immunolabeled for PECAM and LYVE-1 to identify blood and lymphatic endothelial cells, respectively. Tissues selected randomly from those containing vascular networks were quantified for angiogenesis and lymphangiogenesis. Results VEGF-C treatment resulted in an increase in the density of blood vessel sprouting compared to controls by day 3. By day 5, lymphatic sprouting was increased compared to controls. Conclusions These results are consistent with in vivo findings that lymphangiogenesis lags angiogenesis after chronic stimulation and establish a tool for investigating the interrelationships between lymphangiogenesis and angiogenesis in a multisystem microvascular environment.

In pathological scenarios, such as tumor growth and diabetic retinopathy, blocking angiogenesis would be beneficial. In others, such as myocardial infarction and hypertension, promoting angiogenesis might be desirable. Due to their putative influence on endothelial cells, vascular pericytes have become a topic of growing interest and are increasingly being evaluated as a potential target for angioregulatory therapies. For example, the strategy of manipulating pericyte recruitment to capillaries could result in anti- or pro-angiogenic effects. However, our current understanding of pericytes is limited by knowledge gaps regarding pericyte identity and lineage. To use a music analogy, this review is a “mash-up” that attempts to integrate what we know about pericyte functionality and expression with what is beginning to be elucidated regarding their regenerative potential. We explore the lingering questions regarding pericyte phenotypic identity and lineage. The expression of different pericyte markers (e.g., SMA, Desmin, NG2 and PDGFR-β) varies for different subpopulations and tissues. Previous use of these markers to identify pericytes has suggested potential phenotypic overlaps and plasticity toward other cell phenotypes. Our review chronicles the state of the literature, identifies critical unanswered questions, and motivates future research aimed at understanding this intriguing cell type and harnessing its therapeutic potential.

Understanding the mechanisms behind endothelial cell identity is crucial for the goal of manipulating microvascular networks. Lysophosphatidic acid (LPA) and serum stimulation have been suggested to induce a lymphatic identity in blood endothelial cells in vitro. The objective of this study was to determine if LPA or serum induces blood‐to‐lymphatic vessel phenotypic transition in microvascular networks. The rat mesentery culture model was used to observe the effect of stimulation on blood and lymphatic microvascular networks ex vivo. Vascularized mesenteric tissues were harvested from adult Wistar rats and cultured with LPA or 10% serum for up to 5 days. Tissues were then immunolabeled with PECAM to identify blood vessels and LYVE‐1 or Prox1 to identify lymphatic vessels. We show that while LPA caused capillary sprouting and increased vascular length density in adult microvascular networks, LPA did not cause a blood‐to‐lymphatic phenotypic transition. The results suggest that LPA is not sufficient to cause blood endothelial cells to adopt a lymphatic identity in adult microvascular networks. Similarly, serum stimulation caused robust angiogenesis and increased lymphatic/blood vessel connections, yet did not induce a blood‐to‐lymphatic phenotypic transition. Our study highlights an understudied area of lymphatic research and warrants future investigation into the mechanisms responsible for the maintenance of blood and lymphatic vessel identity.

Investigation into the mechanisms driving cancer cell behavior and the subsequent development of novel targeted therapeutics requires comprehensive experimental models that mimic the complexity of the tumor microenvironment. Recently, our laboratories have combined a novel tissue culture model and laser direct-write, a form of bioprinting, to spatially position single or clustered cancer cells onto ex vivo microvascular networks containing blood vessels, lymphatic vessels, and interstitial cell populations. Herein, we highlight this new model as a tool for quantifying cancer cell motility and effects on angiogenesis and lymphangiogenesis in an intact network that matches the complexity of a real tissue. Application of our proposed methodology offers an innovative ex vivo tissue perspective for evaluating the effects of gene expression and targeted molecular therapies on cancer cell migration and invasion. J. Cell. Physiol. 231: 2333-2338, 2016. © 2016 Wiley Periodicals, Inc.

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.

Elevated blood pressure during hypertension has been associated with microvascular rarefaction defined by loss of microvessels. However, whether rarefaction is a result of impaired angiogenesis remains unclear. The objective of this study was to compare angiogenesis across the time course of mesenteric microvascular network remodeling in adult spontaneously hypertensive versus normotensive rats. Angiogenic responses in 15–16-week-old SHR and Wistar rats at 0, 3, 5, 10 or 25 days post 20 minute exteriorization of the mesentery were quantified. Consistent with the phenomenon of rarefaction, vascularized area in unstimulated SHR was decreased compared to Wistar. By 25 days, SHR vascular area had increased to the Wistar level and vascular length density and capillary sprouting were comparable. At 3 and 5 days, SHR and Wistar tissues displayed an increase in the capillary sprouting and vascular density relative to their unstimulated controls. At 10 days, capillary sprouting in the SHR remained elevated. The percent change in vascular density was elevated in the SHR compared to the Wistar group at 3 and 5 days and by 25 days the rate of change was more negative. Our results suggest that SHR networks undergo an increased rate of growth followed by an increased rate of pruning.

Fluid shear stress has been implicated as a regulator of sprouting angiogenesis. However, whether endothelial cells within capillary sprouts in vivo experience physiologically relevant shear stresses remains unclear. The objective of our study is to estimate the shear stress distribution along the length of a capillary sprout through computational modeling of blood flow in a blind-ended channel branching off a host vessel. In this model, we use sprout geometries typical for the rat mesenteric microvasculature and consider three types of boundary conditions: (1) a non-permeable vessel wall, (2) a uniformly permeable vessel wall, and (3) a non-permeable vessel wall with open slots (representative of endothelial clefts). Our numerical simulation predicts that for each boundary condition a local maximum shear stress (13.9, 8.9, and 13.3 dyne cm−2, respectively) occurs at the entrance of a 50 μm long, 6 μm diameter sprout branching at 90° off of a 11 μm diameter host vessel. The shear stress dropped below 0.2 dyne cm−2, a threshold for endothelial cell activation, within 4.1 μm of the entrance for the non-permeable wall case and 4.2 μm for the uniformly permeable wall case. Shear stress magnitudes within the sprout were above 0.2 dyne cm−2 for longer sprout scenarios and peaked at 5.9 dyne cm−2 at endothelial cell clefts. These results provide a first estimate of relative fluid shear stress magnitudes along a capillary sprout and highlight the importance of investigating endothelial cell responses to flow conditions during angiogenesis in tumors and other altered microenvironments.

Angiogenesis and lymphangiogenesis often occur in response to tissue injury or in the presence of pathology (e.g., cancer), and it is these types of environments in which macrophages are activated and increased in number. Moreover, the blood vascular microcirculation and the lymphatic circulation serve as the conduits for entry and exit for monocyte-derived macrophages in nearly every tissue and organ. Macrophages both affect and are affected by the vessels through which they travel. Therefore, it is not surprising that examination of macrophage behaviors in both angiogenesis and lymphangiogenesis has yielded interesting observations that suggest macrophages may be key regulators of these complex growth and remodeling processes. In this review, we will take a closer look at macrophages through the lens of angiogenesis and lymphangiogenesis, examining how their dynamic behaviors may regulate vessel sprouting and function. We present macrophages as a cellular link that spatially and temporally connects angiogenesis with lymphangiogenesis, in both physiological growth and in pathological adaptations, such as tumorigenesis. As such, attempts to therapeutically target macrophages in order to affect these processes may be particularly effective, and studying macrophages in both settings will accelerate the field's understanding of this important cell type in health and disease.

While cancer cell invasion and metastasis are dependent on cancer cell–stroma, cancer cell–blood vessel, and cancer cell–lymphatic vessel interactions, our understanding of these interactions remain largely unknown. A need exists for physiologically-relevant models that more closely mimic the complexity of cancer cell dynamics in a real tissue environment. The objective of this study was to combine laser-based cell printing and tissue culture methods to create a novel ex vivo model in which cancer cell dynamics can be tracked during angiogenesis in an intact microvascular network. Laser direct-write (LDW) was utilized to reproducibly deposit breast cancer cells (MDA-MB-231 and MCF-7) and fibroblasts into spatially-defined patterns on cultured rat mesenteric tissues. In addition, heterogeneous patterns containing co-printed MDA-MB-231/fibroblasts or MDA-MB-231/MCF-7 cells were generated for fibroblast-directed and collective cell invasion models. Printed cells remained viable and the cells retained the ability to proliferate in serum-rich media conditions. Over a culture period of five days, time-lapse imaging confirmed fibroblast and MDA-MB-231 cell migration within the microvascular networks. Confocal microscopy indicated that printed MDA-MB-231 cells infiltrated the tissue thickness and were capable of interacting with endothelial cells. Angiogenic network growth in tissue areas containing printed cancer cells was characterized by significantly increased capillary sprouting compared to control tissue areas containing no printed cells. Our results establish an innovative ex vivo experimental platform that enables time-lapse evaluation of cancer cell dynamics during angiogenesis within a real microvascular network scenario.

The design of therapies aimed at manipulating microvascular growth requires understanding the relationships between angiogenesis and lymphangiogenesis. A critical question remains whether blood and...

New models of angiogenesis that mimic the complexity of real microvascular networks are needed. Recently, our laboratory demonstrated that cultured rat mesentery tissues contain viable microvascular networks and could be used to probe pericyte-endothelial cell interactions. The objective of this study was to demonstrate the efficacy of the rat mesentery culture model for anti-angiogenic drug testing by time-lapse quantification of network growth. Mesenteric windows were harvested from adult rats, secured in place with an insert, and cultured for 3 days according to 3 experimental groups: 1) 10% serum (angiogenesis control), 2) 10% serum + sunitinib (SU11248), and 3) 10% serum + bevacizumab. Labeling with FITC conjugated BSI-lectin on Day 0 and 3 identified endothelial cells along blood and lymphatic microvascular networks. Comparison between day 0 (before) and 3 (after) in networks stimulated by 10% serum demonstrated a dramatic increase in vascular density and capillary sprouting. Growing networks contained proliferating endothelial cells and NG2+ vascular pericytes. Media supplementation with sunitinib (SU11248) or bevacizumab both inhibited the network angiogenic responses. The comparison of the same networks before and after treatment enabled the identification of tissue specific responses. Our results establish, for the first time, the ability to evaluate an anti-angiogenic drug based on time-lapse imaging on an intact microvascular network in an ex vivo scenario.

Recently the authors have shown that neuron-glial antigen 2 (NG2) is expressed by perivascular cells along arterioles and capillaries, but not along venules in quiescent rat mesenteric microvascular networks. To investigate how the spatial distribution of this proteoglycan changes during microvascular remodeling, the objective of this study was to characterize the expression of NG2 in adult rat mesenteric microvascular networks undergoing active remodeling.The distribution of NG2 expression was evaluated in adult rat mesenteric microvascular networks. Tissues were harvested from 250 g, female, Sprague-Dawley rats at 1, 3, and 5 days poststimulation and double immunolabeled for NG2 and CD31 (endothelial cell marker).After 1 day, NG2 expression was observed along 27 +/- 11% of network draining venules (14-55 microm) and after 3 days, 59 +/- 10% of draining venules (13-59 microm) stained positive for the proteoglycan. By 5 days poststimulation, the percentage of network draining venules (18-59 microm) staining positive for NG2 returned to 18 +/- 7%, indicating a downregulation of the proteoglycan toward quiescent levels along larger-sized venules.The results suggest that NG2 proteoglycan expression is transiently upregulated along venules during microvascular remodeling, implicating NG2 as a marker of activated venules.

Tumor growth requires uncontrolled angiogenesis, defined as the growth of new capillaries from existing blood vessels. Developing anti-tumor therapies necessitates understanding how cancer cells an...

Therapies aimed at manipulating the microcirculation require the ability to control angiogenesis, defined as the sprouting of new capillaries from existing vessels. In multiple pathologies (cancer, retinopathies, rheumatoid arthritis), blocking angiogenesis would be beneficial. In others (myocardial infarction, stroke, hypertension), promoting angiogenesis would be desirable. We know vascular pericytes elongate around endothelial cells and are functionally associated with regulating vessel stabilization, vessel diameter, and endothelial cell proliferation. During angiogenesis, bidirectional pericyte-endothelial cell signaling is critical for capillary sprout formation. Observations of pericytes leading capillary sprouts also implicate their role in endothelial cell guidance. As such, pericytes have recently emerged as a therapeutic target to promote or inhibit angiogenesis. Advancing our basic understanding of pericytes and the ability to develop pericyte-related therapies are challenged, like in many other fields, by questions regarding cell identity. This review article discusses what we know about pericyte phenotypes and the opportunity to advance our understanding of pericytes by defining specific pericyte cell populations involved in capillary sprouting.

Objective: Angiogenesis is the growth of new vessels from pre-existing vessels and commonly associated with two modes: capillary sprouting and capillary splitting. Previous work by our laboratory suggests vascular island incorporation might be another endothelial cell dynamic involved in microvascular remodeling. Vascular islands are defined as endothelial cell segments disconnected from nearby networks, but their origin remains unclear. The objective of this study was to determine whether vascular islands associated with microvascular regression are involved in network regrowth. Methods: Mesenteric tissues were harvested from adult male Wistar rats according to the experimental groups: unstimulated, post stimulation (10 and 70 days), and 70 days post stimulation + restimulation (3 and 10 days). Stimulation was induced by mast cell degranulation via intraperitoneal injections of compound 48/80. Tissues were immunolabeled for PECAM (endothelial cells), neuron-glial antigen 2 (NG2) (pericytes), collagen IV (basement membrane), and BrdU (proliferation). Results: Percent vascular area per tissue area and length density increased by day 10 post stimulation compared to the unstimulated group. At day 70, vascular area and length density were then decreased, indicating vascular regression compared to the day 10 levels. The number of vascular islands at day 10 post stimulation was dramatically reduced compared to the unstimulated group. During regression at day 70, the number of islands increased. The disconnected endothelial cells were commonly bridged to surrounding networks by collagen IV labeling. NG2-positive pericytes were observed both along the islands and the collagen IV tracks. At 3 days post restimulation, vascular islands contained BrdU-positive cells. By day 10 post restimulation, when vascular area and length density were again increased, and the number of vascular islands was dramatically reduced. Conclusion: The results suggest that vascular islands originating during microvascular regression are capable of undergoing proliferation and incorporation into nearby networks during network regrowth.

Lymphatic and blood microvascular systems play a coordinated role in the regulation of interstitial fluid balance and immune cell trafficking during inflammation. The objective of this study was to characterize the temporal and spatial relationships between lymphatic and blood vessel growth in the adult rat mesentery following an inflammatory stimulus.Mesenteric tissues were harvested from unstimulated adult male Wistar rats and at 3, 10, and 30 days post compound 48/80 stimulation. Tissues were immunolabeled for PECAM, LYVE-1, Prox1, podoplanin, CD11b, and class III β-tubulin. Vascular area, capillary blind end density, and vascular length density were quantified for each vessel system per time point. Blood vascular area increased compared to unstimulated tissues by day 10 and remained increased at day 30. Following the peak in blood capillary sprouting at day 3, blood vascular area and density increased at day 10. The number of blind-ended lymphatic vessels and lymphatic density did not significantly increase until day 10, and lymphatic vascular area was not increased compared to the unstimulated level until day 30. Lymphangiogenesis correlated with the upregulation of class III β-tubulin expression by endothelial cells along lymphatic blind-ended vessels and increased lymphatic/blood endothelial cell connections. In local tissue regions containing both blood and lymphatic vessels, the presence of lymphatics attenuated blood capillary sprouting.Our work suggests that lymphangiogenesis lags angiogenesis during inflammation and motivates the need for future investigations aimed at understanding lymphatic/blood endothelial cell interactions. The results also indicate that lymphatic endothelial cells undergo phenotypic changes during lymphangiogenesis.

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.

Microvacular network growth and remodeling are critical aspects of wound healing, inflammation, diabetic retinopathy, tumor growth and other disease conditions1, 2. Network growth is commonly attributed to angiogenesis, defined as the growth of new vessels from pre-existing vessels. The angiogenic process is also directly linked to arteriogenesis, defined as the capillary acquisition of a perivascular cell coating and vessel enlargement. Needless to say, angiogenesis is complex and involves multiple players at the cellular and molecular level3. Understanding how a microvascular network grows requires identifying the spatial and temporal dynamics along the hierarchy of a network over the time course of angiogenesis. This information is critical for the development of therapies aimed at manipulating vessel growth. The exteriorization model described in this article represents a simple, reproducible model for stimulating angiogenesis in the rat mesentery. It was adapted from wound-healing models in the rat mesentery4-7, and is an alternative to stimulate angiogenesis in the mesentery via i.p. injections of pro-angiogenic agents8, 9. The exteriorization model is attractive because it requires minimal surgical intervention and produces dramatic, reproducible increases in capillary sprouts, vascular area and vascular density over a relatively short time course in a tissue that allows for the two-dimensional visualization of entire microvascular networks down to single cell level. The stimulated growth reflects natural angiogenic responses in a physiological environment without interference of foreign angiogenic molecules. Using immunohistochemical labeling methods, this model has been proven extremely useful in identifying novel cellular events involved in angiogenesis. Investigators can readily correlate the angiogenic metrics during the time course of remodeling with time specific dynamics, such as cellular phenotypic changes or cellular interactions4, 5, 7, 10, 11.

Background Observations in our laboratory provide evidence of vascular islands, defined as disconnected endothelial cell segments, in the adult microcirculation. The objective of this study was to determine if vascular islands are involved in angiogenesis during microvascular network growth. Results Mesenteric tissues, which allow visualization of entire microvascular networks at a single cell level, were harvested from unstimulated adult male Wistar rats and Wistar rats 3 and 10 days post angiogenesis stimulation by mast cell degranulation with compound 48/80. Tissues were immunolabeled for PECAM and BRDU. Identification of vessel lumens via injection of FITC-dextran confirmed that endothelial cell segments were disconnected from nearby patent networks. Stimulated networks displayed increases in vascular area, length density, and capillary sprouting. On day 3, the percentage of islands with at least one BRDU-positive cell increased compared to the unstimulated level and was equal to the percentage of capillary sprouts with at least one BRDU-positive cell. At day 10, the number of vascular islands per vascular area dramatically decreased compared to unstimulated and day 3 levels. Conclusions These results show that vascular islands have the ability to proliferate and suggest that they are able to incorporate into the microcirculation during the initial stages of microvascular network growth.

An emerging area of microvascular research focuses on the links between neural and vascular patterning. However, the functional dependence between vascular and neural growth in adult tissues remains underinvestigated. The objective of this study was to determine the spatial and temporal coordination between vascular and neural networks over a time course of adult microvascular growth. Mesentery tissues from adult male Wistar rats were harvested prior to stimulation, and 2, 10 and 30 days after angiogenesis stimulated by mast cell degranulation. Tissues were immunolabeled for PECAM (endothelial cell marker) and class III β-tubulin (peripheral nerve marker). Neurovascular alignment was quantified per vessel category: arterioles (>20 µm), pre-capillary arterioles (10–20 µm), post-capillary venules (10–20 µm), venules (>20 µm), capillaries (

A full understanding of the functional role for pericytes in microvascular network growth requires identifying the specific cell phenotypes involved in angiogenesis. The objective of this study was to evaluate class III β-tubulin expression along remodeling adult rat mesenteric microvascular networks. Mesenteric tissues were harvested from unstimulated adult male Wistar rats and at 2, 10 and 30 days post-compound 48/80 stimulation (n=4 per experimental group). Tissues were immunohistochemically labeled with antibodies for class III β-tubulin, NG2 and PECAM. In unstimulated microvascular networks, class III β-tubulin was nerve specific, and did not identify vascular cells along PECAM positive arterioles, venules, and capillaries. Two days post 48/80 stimuli, class III β-tubulin labeling of perivascular cells, including pericytes and smooth muscle cells, was observed along capillary sprouts, capillaries, venules, and arterioles in network regions characterized by increased vessel density and tortuosity. Pericyte identity along capillaries and capillary sprouts was confirmed by cell morphology and co-labeling with NG2. The percentage of vessels with class III β-tubulin positive labeling decreased at subsequent time points and temporally correlated with the time course of capillary sprouting. The results identify class III β-tubulin as a marker of angiogenic perivascular cells and suggest that specific pericyte phenotypes are associated with capillary sprouting.

Microvascular rarefaction, defined by a loss of terminal arterioles, small venules, and/or capillaries, is a common characteristic of the hypertension syndrome. While rarefaction has been associated with vessel-specific free radical production, deficient leukocyte adhesion, and cellular apoptosis, the relationships of rarefaction with structural alterations at the network and cellular level remain largely unexplored. The objective of this study was to examine the architecture and perivascular cell phenotypes along microvascular networks in hypertensive versus normotensive controls in the context of imbalanced angiogenesis. Mesenteric tissues from age-matched adult male spontaneously hypertensive (SHR) and Wistar-Kyoto (WKY) rats were harvested and immunolabeled for PECAM and neuron-glia antigen 2 (NG2). Evaluation of intact rat mesenteric microvascular networks rats suggests that network alterations associated with hypertension are more complex than just a loss of vessels. Typical SHR versus WKY networks demonstrate a reduced branching architecture marked by more proximal arteriole/venous anastomoses and an absence of NG2 labeling along arterioles. Although less frequent, larger SHR microvascular networks display regions of dramatically increased vascular density. SHR and WKY lymphatic networks demonstrate increased vessel diameters and vascular density compared to networks in normotensive Wistar rats (the strain from which both the SHR and WKY originated). These observations provide a rationale for investigating the presence of local angiogenic factors and response of microvascular networks to therapies aimed at reversing rarefaction in genetic hypertension.

The objective of this study was to quantify the proliferation of existing vascular and perivascular cells during a specific form of microvascular remodeling characterized by increased coverage by smooth muscle cells (SMCs), in response to increased mechanical stress. Coordinated ligations of artery/vein pairs in the rat mesentery resulted in hemodynamic stress elevations within the targeted microvascular network. BRDU incorporation per unit length of smooth muscle (SM) alpha-actin positive vessel was evaluated following ligation at 2, 5, and 10 days. At 2 days, BRDU incorporation was significantly increased for both sham and ligated treatments, but the ligated response was not elevated over the sham response. After 5 days, proliferation for both groups returned to unstimulated levels. The results indicate that moderate elevations in hemodynamic stress do not cause perivascular cell proliferation along rat mesenteric microvessels, therefore, the increased coverage of differentiated SMCs along the same microvessels does not involve proliferation of vascular or perivascular cells.

Recent evidence implicating leukocytes in angiogenesis raises the question of whether leukocytes and other cells circulating with the blood in microvascular networks can home to capillary sprouts intraluminally. This study describes an investigation of leukocyte trafficking in sprouting capillaries fabricated using soft lithography. The leukocytes passing with whole blood through existing capillaries were able to enter microfabricated capillary sprouts of variable length and sprouting angle due to the mechanical interaction with red blood cells (RBCs) at the sprouting bifurcation, in spite of the complete absence of blood flow through the blind-ended sprouts or any chemoattractants. The RBCs formed "comet tails" (the densely packed cellular trains forming behind leukocytes as they move through narrow capillaries) and effectively pushed leukocytes into the microfabricated sprouts while bypassing them at the sprouting bifurcation. Individual sprouts filled with several leukocytes, as wells as RBCs and platelets, were observed. The results of this study suggest that (i) blood cells are likely present in capillary sprouts throughout their development, (ii) leukocytes and other circulating cells may use this mechanism to home to capillary sprouts intraluminally for direct engraftment, and (iii) tissues may use this phenomenon as another mechanism for local recruitment of leukocytes from the blood stream.

Analyses of microvascular networks with traditional tracer filling techniques suggest that the blood and lymphatic systems are distinct without direct communications, yet involvement of common growth factors during angiogenesis and lymphangiogenesis suggest that interactions at the capillary level are possible. In order to investigate the structural basis for lymphatic/blood endothelial cell connections during normal physiological growth, the objective of this study was to characterize the spatial relations between lymphatic and blood capillaries in adult rat mesenteric tissue. Using immunohistochemical methods, adult male Wistar rat mesenteric tissues were labeled with antibodies against PECAM (an endothelial marker) and LYVE-1, Prox-1, or Podoplanin (lymphatic endothelial markers) or NG2 (a pericyte marker). Positive PECAM labeling identified apparent lymphatic/blood endothelial cell connections at the capillary level characterized by direct contact or direct alignment with one another. In PECAM labeled networks, a subset of the lymphatic and blood capillary blind ends were connected with each other. Intravital imaging of FITC-Albumin injected through the femoral vein did not identify lymphatic vessels. At contact sites, lymphatic endothelial markers did not extend along blood capillary segments. However, PECAM positive lymphatic sprouts, structurally similar to blood capillary sprouts, lacked observable lymphatic marker labeling. These observations suggest that non-lumenal lymphatic/blood endothelial cell interactions exist in unstimulated adult microvascular networks and highlight the potential for lymphatic/blood endothelial cell plasticity.