This is observed in the Boyden chamber assay but is more accurately measured using phagokinetic track assay methods. This uses colloidal gold-plated coverslips to serve as a substrate for the movement of cells, which displace the colloidal gold leaving a track that can be measured for directional properties and total area Zetter This assay has been modified to permit large-scale screening using beads attached to the bottom of well plates. Similarly, only a small number of endothelial cells are studied in each case, and the assays are time consuming to analyse and interpret.
An alternative type of migration assay is based on the idea that endothelial cell migration into a denuded area is a pivotal event in wound healing in vivo. Endothelial cell monolayers are prepared and permitted to reach confluence. Using a scraping tool, a portion of the monolayer is then cleared of endothelial cells, providing a margin from which endothelial cells migrate to fill the denuded region.
The rate and extent of endothelial cell migration is then monitored microscopically Pepper et al. Quantification is arbitrary, and problems are found with requirements for the running of control and experimental groups under identical growth conditions of confluence, and the denuded area must be precise Auerbach et al. A closer approximation to in vivo endothelial cell migration could possibly be achieved by combining organ culture techniques with cell migration analysis.
An example of this approach is the aortic ring assay, where segments of rat aorta are explanted into collagen or fibrin gels to achieve an environment that permits three-dimensional 3D growth and migration of cells out from the aorta Nicosia et al. However, in this and similar systems, migration is not distinguishable from differentiation, as both occur simultaneously, with outgrowths appearing as capillaries, potentially containing a lumen. Therefore, this assay is more accurately described as a differentiation assay or organ culture assay and, as such, will be described in more detail in the next section.
Assays that stimulate the formation of capillary-like tubules by endothelial cells have become increasingly popular in recent years. The formation of tight junctions between the endothelial cells has been confirmed by electron microscopy Auerbach et al. Recently, a second form of Matrigel has been developed called growth factor-reduced Matrigel in which the levels of stimulatory cytokines and growth factors have been markedly reduced. This more defined preparation avoids problems associated with the gross stimulation of endothelial cells that occurs in standard Matrigel.
However, although Matrigel has been shown to direct endothelial cells along the differentiation pathway Fawcett , it is questionable as to whether the tubes formed on Matrigel possess a lumen. Lumen formation may also not occur when tubules form on collagen or fibrin Zimrin et al.
Recently Sanz et al. This was achieved by scaling down the assay for use in well and well high-density formats where the endothelial cells yielded highly reproducible tubule formation per well. A computer-assisted integrated platform for capturing and processing images of complete wells was then used. This system evaluated the total number of nodes, connected and unconnected tubes as well as their lengths, and was tested in a double-blind experiment, which validated the results Sanz et al. This procedure has eliminated some of the problems of the Matrigel assays with respect to reproducibility and to the difficulties in analysing the tubule formation seen in a total well accurately and has resulted in an easy computerized system for analysing tubule formation.
However, the lack of a lumen and the homogeneous nature of the tubules remain as limitations to this assay protocol. It is also possible to use Matrigel and, more commonly, fibrin clots to represent a 3D angiogenesis system. In this assay, the endothelial cells are sandwiched between layers of matrix either fibrin or Matrigel and then allowed to form tubules over an extended period of time.
Initially, endothelial cells form tubules in the horizontal plane, but over a period of 12 or more days, the endothelial tubes begin to branch upwards and penetrate the gel to form a 3D network of tubules Gagnon et al. Although these assays more closely mimic the in vivo situation, in which endothelial cells do not just form capillaries in two dimensions, quantification of cell behaviour in three dimensions, which is notoriously difficult to analyse, remains a challenge.
The analysis involves taking pictures at different heights e. The length both width, for those in the horizontal plane, and height, for those in the vertical plane and largest diameter of each vessel are measured Gagnon et al. The microvessel density can also be calculated using the Chalkley grid method where an eyepiece-mounted, point Chalkley array graticule is used to assess the vascular density, by counting the number of points on the Chalkley grid coinciding with tubules at different heights through the gel adapted from Fox et al.
The limitations are obvious with only a proportion of tubules and the area or volume of the gel being analysed. Another form of tubule assay involves the coculture of endothelial cells with stromal cells, with or without the provision of an extracellular matrix Montesano et al. One such assay involves the coculture of human fibroblasts and endothelial cells and relies on fibroblasts secreting the necessary matrix components that act as a scaffold for tubule formation Bishop et al.
This assay has been shown to produce tubules that contain lumen, and a more heterogeneous pattern of tubule lengths with some longer tubules than in the Matrigel assays, which more closely resemble the capillary bed in vivo Bishop et al. However, this assay takes 12—14 days and is therefore more time consuming than the Matrigel assay typically 4—24 h , and is a less well-characterized assay as the matrix components secreted by the fibroblasts have not been defined. Also, undefined interactions between endothelial cells and the fibroblasts may occur, making the effect s of drugs or other compounds specifically on endothelial cells hard to identify.
As angiogenesis does not involve endothelial cells alone, studies in recent years have attempted to assess angiogenesis in whole or partial organ culture, including assays such as the rat aortic ring, the chick aortic arch, porcine carotid artery, placental vein disk and fetal mouse bone explant. These assays are all similar, in that segments, disks or sections of the relevant material are cultured in vivo , often in a matrix such as fibrin, and over a period of 10—14 days, the outgrowth of endothelial and other cells is monitored Figure 3.
Quantification is achieved by measuring the number and length of microvessel outgrowths from the primary explant Nicosia et al. However, quantification is a challenge as individual outgrowths are clustered together, and hence area covered by outgrowth is more commonly measured. The model is not truly representative of the microvascular environment encountered in tumour growth as the large number of different factors released by tumour cells and the tumour cells themselves are not present Auerbach et al.
Formation of tubules from an aortic ring.
Current methods for assaying angiogenesis in vitro and in vivo
The aortic ring is cultured in full growth medium containing vascular endothelial growth factor. The appearance of tubules can clearly be seen. These explant assays are considered to come closest to mimicking the in vivo situation because they include the surrounding nonendothelial cells such as smooth muscle cells and pericytes and a supporting matrix. In addition, the endothelial cells are not proliferating at the time of explantation and thus are more representative of the situation found in vivo where angiogenesis is triggered and quiescent endothelial cells respond by becoming proliferative, migrating out from the existing vessels and differentiating into tubules.
Although the aortic ring assay is the most commonly used organ culture assay in angiogenesis research Auerbach et al. Also, many workers report significant variation in the vessel outgrowth between explants from different animals, making the use of large number of donor rats necessary.
Some consider that the porcine carotid artery explant assay is an improvement on the rat aortic ring assay, in that the larger blood vessel derived from the pig enables the running of a number of assays from a single carotid artery, thus eliminating statistical variation between animals Stiffey-Wilusz et al. However, this has obvious drawbacks as the single donor used may not be representative of the true population and, again, a large rather than small vessel is used.
Angiogenesis: In Vivo Systems, Part B, Volume 445 (Methods in Enzymology)
One group has used human placental vein disks to eliminate this issue Jung et al. Another group used day-old fetal mouse metatarsals. As these are still undergoing development, they contain endothelial cells in the perichondrium. When cultured in medium containing fetal calf serum, dissected metatarsals show outgrowth of tube-like structures after 10 days of culture, which stain positively for endothelial cell markers Deckers et al.
These have the advantage of being microvascular in origin, thus more closely representing real angiogenesis, but as the tissues used are from growing embryos, they will be undergoing proliferation before explantation and therefore are not truly representative of the stimulation of nonproliferative endothelial cells that is seen in vivo. A modification of the rat aortic ring assay is the chick aortic arch assay Muthukkaruppan et al.
Also, unlike the adult aorta, embryonic arch endothlelial cells share many properties with microvascular endothlelial cells. However, like the fetal mouse metatarsal assay, these aortic arches are derived from growing embryos. The previous sections discuss the wide array of in vitro procedures now being used to study various aspects of angiogenesis Cockerill et al. However, comparison between different in vitro systems is often difficult, due to differences in the origin of endothelial cells as well as their passage numbers and the composition of different media used.
Studies have shown that a compound affecting cell proliferation, migration or differentiation in vitro may not necessarily regulate endothelial cell activity in vivo Liekens et al. Thus, the in vivo evaluation of angiogenesis-modulating agents is a vital next step in drug development.
A number of in vivo assays have been reviewed previously Jain et al. In the case of the sponge method, the test substance is either directly injected into the sponge Hu et al. Neovascularization can then be assessed by a variety of methods including immunohistological staining e. However, differences in sponge size, shape and composition make comparisons between different studies difficult.
Furthermore, implantation can cause nonspecific immune responses that may themselves lead to an angiogenic response Dellian et al. The test substance can be suspended in the gel, which is then injected subcutaneously where it forms a solid plug allowing the slow release of the substance Passaniti et al. The angiogenic response can then be quantified by the extent of CDpositive vessel growth into the plug or by measuring the haemoglobin content of the plug.
Matrigel is expensive and its analysis is time consuming, but unlike sponges, it is not an artificial matrix and hence provides a more natural environment in which to initiate an angiogenic response. The chick chorioallantoic membrane CAM assay is probably the most widely used in vivo assay for studying angiogenesis Nguyen et al.
The test substance is prepared either in slow-release polymer pellets, absorbed by gelatin sponges, or air-dried onto plastic discs; these are then implanted onto the CAM through a window cut carefully in the eggshell. The lack of a mature, immune system in 7—8-day old chick embryos allows for the study of tumour-induced angiogenesis Folkman The angiogenic effects can be measured by counting the number of blood vessels in a given area using a stereomicroscope. In a variation of the CAM assay, shell-less embryos are cultured in Petri dishes prior to applying the test substance Auerbach et al.
The development of new vasculature in the chick chorioallantoic membrane CAM assay. The appearance of the CAM in the absence a or presence b of thymidine phosphorylase. The CAM assay is a relatively simple and inexpensive in vivo assay suitable for large-scale screening. However, the CAM itself has a well-developed vascular network, thus making it difficult to distinguish new capillaries from existing ones.
It is usually necessary to wait for 3 days after making the window before adding the test substance, to check for any immune response. Finally, the membrane is very sensitive to changes in oxygen tension, making the sealing of the window a vital part of the procedure Auerbach et al. The cornea is an avascular site, and thus, any vessels penetrating from the limbus into the corneal stroma can be identified as newly formed Gimbrone et al.
An angiogenic response can then be initiated by implantation of a slow-release pellet or polymer containing the angiogenic substance Hartwell et al. If the animal is then administered with a test compound, the vascular response can be quantified by computer image analysis following perfusion of the cornea with India ink. This assay is reliable, but compared to the CAM assay, is more expensive and technically demanding, making it impractical for large-scale screening. Furthermore, although the use of rats and mice make the assay cheaper and increases the number of tests that can be performed, the surgery becomes more difficult as eye size decreases.
Finally, scientists face ethical problem when using an assay that involves a major sensory organ. The dorsal air sac model is used to examine the in vivo effect s of substances against the angiogenic response triggered by cancer cells Oikawa et al. Briefly, both sides of a Millipore ring are covered with filters 0. Following treatment with the compound of interest, the chamber is carefully removed and rings of the same diameter placed directly upon the sites that were exposed to a direct contact with the chamber. The angiogenic response can then be assessed by counting the number of newly formed blood vessels that lie within the area marked by the ring, using a dissecting microscope.
This assay is relatively simple to perform, although care must be taken not to irritate the surface upon which the chamber is placed, as this may itself induce angiogenesis and hence mask those blood vessels formed due to the presence of the tumour cells. The in vivo study of chronic angiogenesis has been greatly advanced by the development of several types of transparent chamber, such as the rabbit ear chamber, dorsal skinfold chamber and cranial window chamber. In these systems, a piece of skin ear and skinfold chambers or part of the skull cranial window chamber is removed from an anaesthetized animal.
Tumour cells, or a gel containing angiogenic factors, is then placed on the exposed surface and covered by glass, which is then secured in place; once the animals have recovered, these models allow for the continuous measurement of various parameters in living animals, including gene expression, angiogenesis, pH and blood flow Jain , and hence aid the study of the effect of tissue microenvironment on angiogenesis. The dorsal skin chamber shown in Figure 5 has been developed for rats and mice and has been used to show the importance of angiogenesis to tumour growth, including xenografts in immuno-deficient rodents Leunig et al.
Quantification of results from a chamber assay involves microscopy. The animal is restrained, with the window in the correct position for direct observation; areas of interest are then recorded on to videotape and analysed off-line, either by computer image analysis using image pro plus or capiscope software or manually by on-screen measurement. The dorsal skinfold window chamber model.
After recovery from surgery, the neovascularization in the chamber is imaged using in vivo microscopy. Scale bar, 15 mm. Chamber assays allow for the determination of 3D vessel growth in one animal, typically over a period of 1—3 weeks. As a result, separate groups of mice are not required at each measurement point, and hence, the number of animals used is minimized.
The cranial window chamber assay has several other advantages including better transplantability and induction of a rapid angiogenic response Yuan et al. However, all chamber assays are invasive and technically demanding. The rabbit ear chamber is expensive for routine use, and a period of 4—6 weeks must pass after surgery before the test substance can be placed into the chamber. Let us wish you a happy birthday! Day 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 Month January February March April May June July August September October November December Year Please fill in a complete birthday Enter a valid birthday.
Skin care Face Body. What happens when I have an item in my cart but it is less than the eligibility threshold? Should I pay a subscription fee to always have free shipping? By understanding how bone marrow-derived cells contribute to tumor growth, it may be possible to develop new approaches to cancer therapy.
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- Chapter 15 Methods to Study Myeloid Cell Roles in Angiogenesis - Dimensions.
In this chapter, we discuss experimental methods to examine the roles of myeloid cells in tumor growth and angiogenesis, including methods to identify, isolate, purify, and characterize bone marrow-derived monocytes. We also outline methods to analyze the in vivo roles of myeloid cells in tumor growth and angiogenesis using adoptive transfer, bone marrow transplantation, tumor models and immunohistochemistry for markers of vessels and myeloid cells. Finally, we review methods to characterize myeloid cell trafficking in vitro and in vivo.
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