Intact Microvascular Networks Research Articles

Time-Lapse Observation of Cell Dynamics During Angiogenesis Using the Rat Mesentery Culture Model.

The ability to track cells and their interactions with other cells during physiological processes offers a powerful tool for scientific discovery. An ex vivo model that enables real-time investigation of cell migration during angiogenesis in adult microvascular networks would enable observation of endothelial cell dynamics during capillary sprouting. Angiogenesis is defined as the growth of new blood vessels from existing ones and involves multiple cell types including endothelial cells, pericytes, and interstitial cells. The incorporation of these cell types in a physiologically relevant environment, however, represents a challenge for biomimetic model development. Recently, our laboratory has developed the rat mesentery culture model, which enables investigation of angiogenesis in an intact tissue. The objective of this chapter is to detail a protocol for tracking cellular dynamics during angiogenesis using the rat mesentery tissue culture model. The method involves harvesting mesentery tissues from adult SD-EGFP rats, culturing them in MEM+10% fetal bovine serum, and imaging network regions over the time course of angiogenesis. In example applications, time-lapse comparison of microvascular networks in cultured tissues confirmed dramatic increases in GFP-positive capillary sprouting and GFP-positive segment density. Additionally, tracking of individual capillary sprout extensions revealed their ability to "jump" by disconnecting from one vessel segment and reconnecting to another segment in the network. GFP-positive sprouts were also capable of undergoing subsequent regression. The representative results support the use of the rat mesentery culture model for identifying and tracking cellular dynamics during angiogenesis in intact microvascular networks.

Bioprinting on Live Tissue for Investigating Cancer Cell Dynamics.

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.

Effective Reinnervation of Skeletal Muscle is Impaired by Disrupting Microvascular Regeneration Following Acute Injury

Acute injury of skeletal muscle damages myofibers, microvessels and neuromuscular junctions (NMJs). Although myofiber regeneration is well‐characterized, little is known of concurrent events in microvessels and motor nerves during skeletal muscle regeneration. Because growth and patterning are coordinated between nerves and microvessels during development, we tested the hypothesis that effective microvascular regeneration is required for restoring neuromuscular structure and function post injury in the adult. To impair angiogenesis, mice were bred for endothelial cell (EC)‐specific deletion of ephrin‐B2. Male mice (3–6 months old; n=2–3/group) were injected with tamoxifen to delete ephrin‐B2 in ECs (EfnB2CKO; confirmed with PCR); uninjected mice of the same genotype were used as controls. The gluteus maximus (GM) or tibialis anterior (TA) muscle was injured by local injection of 1.2% BaCl2 and muscles were evaluated at 10 days post injury, by which time morphological and functional myofiber regeneration will have occurred. Intravascular injection of fluorescent wheat germ agglutinin (WGA; stains only perfused vessels) or immunolabeling for CD31 (stains all ECs) revealed microvessels of the GM in EfnB2CKO to be vacuolated with disorganized ECs vs. intact microvascular networks with aligned ECs in control mice. Using indirect stimulation via the sciatic nerve, isometric force production in the TA was 19±6 N/cm2 in EfnB2CKO vs. 33±3 N/cm2 in control mice; force produced with direct electrical field stimulation was similar between groups. Immunostaining for neurofilament‐heavy revealed reduced presynaptic coverage of NMJs in EfnB2CKO vs. control [60±9% vs. 74±10% of acetylcholine receptors (AChRs) per 200 μm2 NMJ area]; postsynaptic staining with α‐bungarotoxin revealed dispersed AChRs [200±118 vs. 92±43 fragments/200 μm2, respectively]. These preliminary findings indicate that the loss of ephrin‐B2 in ECs results in functional deficits for neuromuscular transmission and NMJ morphology in association with impaired microvascular regeneration following acute injury. We conclude that regeneration of intact NMJs with functional reinnervation of myofibers requires effective revascularization and recovery of the microcirculation following skeletal muscle injury.Support or Funding InformationSupport: APS Postdoctoral Fellowship (ABM), R01AR067450 (DDWC), R37HL041026 (SSS)

A novel tissue culture model for evaluating the effect of aging on stem cell fate in adult microvascular networks.

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 5days 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.

Investigating Pericyte Dynamics during Angiogenesis in the Mouse Mesometrium Culture Model

Angiogenesis requires multiple cells and systems working in concert to develop new blood vessels from existing ones. Hence, understanding the exact dynamics between cells and systems proves to be beneficial when developing innovative therapies. In the microcirculation, endothelial cells that line the blood vessels are supported by perivascular cells that are elongated cells called pericytes. Cell membrane proteins that modulate cellular responses of different growth factors play an important role during microvascular remodeling. For pericytes, the neural glial antigen‐2 (NG2) proteoglycan is a cell membrane protein that is exclusively expressed by mural cells in mouse microvasculature. Pericytes' multifaceted roles in angiogenesis and where they originate still requires further research. Our lab has recently introduced the mouse mesometrium culture model that allows for the investigation of cell dynamics within intact microvascular networks. The objective of this study was to demonstrate the usefulness of the mouse mesometrium culture model for investigating NG2 inhibition and cell‐lineage tracing. For the NG2 inhibition studies, female C57BL/6 (WT) and NG2 KO were euthanized to aseptically harvest mesometrium tissues and culture them in MEM + 1% PS and 20% FBS at standard incubating conditions. WT and NG2 KO tissues were cultured for 3 days before labeling with PECAM and NG2 antibodies to quantify angiogenesis. PECAM and NG2 labeling confirmed the lack of NG2‐positive pericytes on capillaries from NG2 KO tissues. The angiogenic response after culture from NG2 KO tissues was significantly decreased when compared to WT tissues (NG2 KO: 0.98 ± 0.29; WT: 2.11 ± 0.32 number of sprouts per total vessel length, p = 0.019). For cell‐lineage tracing studies, female NG2dsRed mice were given a daily IP injection of 0.1 mL of sunflower oil for 5 consecutive days to increase microvascular density. Mesometrium tissues were harvested 21 days post last injection and cultured in MEM + 1% PS and 20% FBS at standard incubating conditions for 5 days. The tissues were imaged for time‐lapse at least every 12 hours to track individual cell interactions. Time‐lapse imaging taken every 2 hours for 12 hours on days 1–3 of NG2dsRed tissues revealed that NG2‐positive pericytes migrated along pre‐existing vessels to new vessel segments. Moreover, a subpopulation of NG2‐positive interstitial cells were observed to migrate throughout the tissue. These results validate that inhibiting NG2 expression has a direct effect on angiogenesis in the mouse mesometrium culture model. Furthermore, it highlights the usage of the mouse mesometrium culture model to track individual cell‐to‐cell dynamics of pericytes in intact microvasculature during angiogenesis.Support or Funding InformationNIH R01AG049821This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

A Novel ex vivo Mouse Mesometrium Culture Model for Investigating Angiogenesis in Microvascular Networks

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.

Endothelial Cell Phenotypes are Maintained During Angiogenesis in Cultured Microvascular Networks

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.

An Ex Vivo Platform for Studying Angiogenesis in Perfused Microvascular Networks

A challenge in tissue engineering biomimetic models for studying angiogenesis is building the physiological complexity of real microvascular networks. Utilizing an alternative top‐down approach, our laboratory recently 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. However, a current limitation of the model is that microvessels are not perfused. The objective of this study was to demonstrate the feasibility of perfusing microvascular networks during the culture of rat mesentery tissue. Adult Wistar rat mesentery tissues were harvested along with a feeding second order arteriole and venule, and then transferred to a customized tissue chamber for perfusion. The arteriole was cannulated with a glass pipette (40 – 60 μm diameter) and secured in place with 7–0 suture. Injection with blue food dye through the cannulated arteriole identified the lumens of vessels across the hierarchy of intact microvascular networks and confirmed successful perfusion. Tissues with cannulated arterioles were cultured in the perfusion chamber up to three days with either minimum essential media (MEM) or MEM supplemented with 10% serum. Live/dead labeling confirmed that cannulated mesentery tissues remain viable after three days in culture. Additional labeling with BSI‐lectin identified endothelial cells along microvascular networks and confirmed the tissues were undergoing angiogenesis, characterized by increases in vascular density and capillary sprouting. These results demonstrate the ability to perfuse intact microvascular networks ex vivo in cultured mesentery tissues and supports the novelty of the rat mesentery culture model as a biomimetic tool for investigating angiogenesis.Support or Funding InformationNIH R01AG049821This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Evaluation of Arteriolar Smooth Muscle Cell Function in an Ex Vivo Microvascular Network Model

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.

Development of the Ex Vivo Mouse Mesometrium Model to Investigate Multicellular Dynamics During Angiogenesis

The development of biomimetic in vitro or ex vivo models that can incorporate the complexity of intact microvascular networks would enable the investigation of multicellular dynamics associated with angiogenesis and novel experimental platforms for pre‐clinical therapy testing. Recently, our laboratory has introduced the rat mesentery culture model as such a tool. The applicability of this model would be enhanced with the use of murine tissue, yet unfortunately, the mouse mesentery is avascular. A potential analog to the mesentery is the mesometrium, the connective tissue of the uterine horns. The objective of this study was to demonstrate that the mouse mesometrium contains intact microvascular networks that can be cultured to investigate multicellular dynamics during angiogenesis. Mesometrium windows, defined as the translucent connective tissues attached to the uterine horns, were harvested from adult C57Bl/6 female mice and cultured in minimum essential media with 20% fetal bovine serum for 3 and 5 days. The tissues were immunohistochemically labeled with platelet endothelial cell adhesion molecule (PECAM), neuron‐glial antigen 2 (NG2), and alpha‐smooth muscle actin (α‐SMA) to identify endothelial cells, pericytes, and smooth muscle cells, respectively. The mesometrium proved to have an intact microvascular network with arterioles, venules and capillaries. Arterioles and venules were differentiated by the PECAM‐positive cell morphology, and the α‐SMA‐positive cell morphology that wraps the vessels. α‐SMA and NG2 labeling confirmed the presence of smooth muscle cells along the larger vessels and pericytes along capillaries. After 5 days in culture, tissues remained viable as confirmed with a LIVE/DEAD assay. Compared to day 0, uncultured tissues, capillary sprouts per venule length was dramatically increased by day 3 and day 5 (Day 0: 0.09 ± 0.02; Day 3: 4.10 ± 0.75; Day 5: 4.37 ± 0.60 spouts/mm). For day 3 and day 5, capillary sprouting preferentially occurred of venules versus arterioles. Furthermore, while the number of capillary sprouts were comparable for day 3 and day 5, sprouts appeared to be longer on day 5. Capillary sprouts were also observed to contain NG2‐positive, and SMA‐negative pericytes. These results introduce the mouse mesometrium culture model as a novel ex vivo approach for elucidating pericyte‐endothelial cell dynamics associated with capillary sprouting at specific vessel locations in a real microvascular network scenario.Support or Funding InformationNIH R01AG049821, NIH P20GM103629

An Ex Vivo Method for Time-Lapse Imaging of Cultured Rat Mesenteric Microvascular Networks.

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.

An &lt;em&gt;Ex Vivo&lt;/em&gt; Method for Time-Lapse Imaging of Cultured Rat Mesenteric Microvascular Networks

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.

An Ex Vivo Tissue Culture Model for Anti-angiogenic Drug Testing.

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.

Lysophosphatidic Acid Stimulation Does Not Induce a Lymphatic Identity along Blood Vessels in Intact Microvascular Networks Ex Vivo

The design of therapies aimed at manipulating microvascular growth requires understanding the relationships between angiogenesis and lymphangiogenesis. A critical question remains whether blood and lymphatic vessels can switch their identity in adult tissues. Lysophosphatidic acid (LPA), a lipid linked to oncogenesis, wound healing, and inflammation, has been shown to upregulate lymphatic marker expression by HUVECs in vitro. The objective of this study was to determine if LPA stimulation induces blood to lymphatic vessel phenotypic transition in intact microvascular networks. Using the rat mesentery culture model, which enables ex vivo investigation of angiogenesis and lymphangiogenesis, tissues from adult male Wistar rats were cultured for 3 days in serum‐free minimum essential media (MEM) or MEM with LPA (20 µM). Tissues were labeled for PECAM and either LYVE‐1, Prox1, or podoplanin to identify blood and lymphatic vessels, respectively. LPA stimulation caused a significant increase in blood capillary sprouting indicative of angiogenesis. Observation of LYVE‐1+ filopodia extensions suggests LPA stimulation also causes lymphangiogenesis. However, no indication of blood‐to‐lymphatic phenotype transition was observed. Blood vessels identified based on morphology and PECAM labeling intensity lacked lymphatic marker expression. These results indicate LPA stimulation is not sufficient to induce a lymphatic phenotype in blood vessels of intact microvascular networks. This emphasizes the importance of investigating lymphatic/blood vessel relationships in real microvascular network versus cell culture scenarios and motivates new questions regarding the mechanisms that maintain vessel identity.

Tracking Human Adipose‐Derived Stem Cells (hASCs) in an Ex Vivo Microvascular Network Model

Models that mimic angiogenesis are valuable for the investigation of underlying mechanisms and the pre-clinical development of therapies. We have recently demonstrated that the rat mesentery culture model offers the potential for time-lapse investigation of mechanistic cell–cell interactions at specific locations across intact blood and lymphatic microvascular networks ex vivo. The objective of this study was to demonstrate the capability of the rat mesentery culture model to track exogenously delivered human adipose-derived stem cell (hASC) migration and differentiation dynamics within an intact microvascular network. RFP-transfected hASCs were seeded onto the harvested adult Wistar rat mesenteric tissues and cultured for 3 or 5 days. In separate studies, RFP-transfected hASCs were pre-positioned in “cell spots” (~40 cells per spot) via laser direct write cell printing on the tissues and cultured up to 5 days. Lectin labeling of endothelial cells on day 0 and later time points enabled time-lapse imaging of cells during angiogenesis. For both cell delivery approaches, cells integrated within the tissues and displayed elongated morphologies. A subset of hASCs adopted a perivascular location typical of vascular pericytes. The laser direct write printing studies confirmed cell migration. Our results establish an ex vivo model for evaluating the initial differentiation, growth and migration dynamics of stem cells in an intact microvascular network scenario.

An ex vivo model for anti-angiogenic drug testing on intact microvascular networks.

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.

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

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.

VEGF‐C Induces Lymphangiogenesis and Angiogenesis in the Rat Mesentery Culture Model

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. 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. 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. 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.

The effect of cancer cells on angiogenesis: engineering the cancer niche (676.13)

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 and their microenvironment synergistically influence capillary sprouting from existing vascular networks. Recently, we demonstrated the use of the rat mesentery culture model for investigating angiogenesis in an intact microvascular network scenario. The objective of this study was to demonstrate the use of this model to determine the effects of cancer cells on angiogenesis. Mesenteric tissue windows were harvested from adult male Wistar rats and cultured for 5 days according to the following experimental groups: 1) Control (MEM + 10% DMEM + 1% FBS; n = 7 tissues) and 2) Cancer Cell (MEM + 10% DMEM + 1% FBS + cells; n = 6 tissues). For the cancer cell group, 2x10^5 RFP‐transfected metastatic MDA‐MB‐231 breast cancer cells were seeded onto tissues on day 0. On day 5, cancer cells remained viable, as confirmed by live/dead assay. More importantly, cells were observed to be in the same focal plane and region as vessels along intact microvascular networks. The presence of cancer cells decreased vascular length density and the number of capillary sprouts per vascular area compared to the Control (no cell) group. Our results suggest that cancer cells alone are not sufficient to induce angiogenesis. Application of the rat mesentery culture model as a multicellular breadboard system to engineer the cancer cell/vascular niche will offer a new tool for tumor angiogenesis research.Grant Funding Source: Supported by Tulane Center for Aging and NIH 5‐P20GM103629‐02

A novel ex vivo tissue culture assay for determining the effects of anti‐tumor drugs on angiogenesis (676.14)

Tumor growth is highly dependent upon angiogenesis and the interplay between the tumor stroma and the microvasculature. A challenge in evaluating anti‐tumor therapies is the inability to decouple treatment effects on cancer cells versus the microvascular network. Recently, our laboratory demonstrated that the “rat mesentery culture” model could be used for time‐lapse comparison of intact microvascular networks before and after angiogenesis induction. The objective of this study was to demonstrate that this model could be used to evaluate the anti‐angiogenic effects for a given drug treatment. Mesenteric windows were harvested from adult male Wistar rats and cultured for 3 days in serum containing medium (MEM + 10% Fetal Bovine Serum). Explants were exposed to increasing concentrations of Nelfinavir (Viracept™) (0 ‐ 4.5 μM) and Curcumin (0 ‐ 4.5 μM). Nelfinavir and Curcumin were selected for the study because Nelfinavir causes endothelial dysfunction and Curcumin, an anti‐oxidant and NF‐κB inhibitory dietary supplement, is known to have anti‐angiogenic effects. Preliminary studies also suggest that the combination of Nelfinavir/Curcumin produce synergistic anti‐cancer effects. Labeling tissues with FITC conjugated BSI‐lectin on Day 0 and Day 3 identified endothelial cells along all vessels. Results showed significant dose‐responsive effects of Nelfinavir/Curcumin combinations in suppressing both vessel segment density and the number of capillary sprouts per vascular area. Our findings establish the rat mesentery culture model as a valuable ex vivo tool for the rapid and reproducible screening of drug effects on angiogenesis in an intact network scenario.Grant Funding Source: Supported by Tulane Center for Aging and NIH 5‐P20GM103629‐02