New blood vessel formation follows in principle four cardinal steps: First step is that endothelial cells need to adhere, proliferate and permeabilize the environmental boundaries. Therefore endothelial cells undergo cell division and start to secret proteases to degrade the matrix and brake through the basal lamina, which is the second inner layer of the vessel. Endothelial cells use for instance integrins to travel in tandem.
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This event is connected with the sprouting of newly formed vessel barbs. As a source, angiogenic stimuli can be secreted by activated lymphocytes, tumor cells, microglia and macrophages. Thirdly, endothelial vessel sprouts form new vessels and recruit cellular components to line up a fully functional vessel. The fourth key step is the modeling and reorganization process. Newly formed vessels can maturate or undergo regression.
For each of the four key steps several in vitro assays have been developed. In the past we considered the vascular endothelium as a passive structure which acts like a filter between the blood in the vessel lumen and the vessel wall itself. In fact endothelial cells are active members of the vascular homeostasis playing a vital role in the coagulation and the fibrinolysis system as well as in adhesion and aggregation of blood platelets via secreted activators and inhibitors.
These processes among others can be studied in vitro using human vascular endothelial cells isolated from umbilical veins. Tube formation assay in time lapse modus.
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The left picture is taken 2 h after plating and shows beginning tube formation. Images are given as x10 magnification.
Jaffe in [ 69 ]. Endothelial cells of all origin seem to have the ability to form three-dimensional structures tubes, cobblestone pattern , which can be fostered by coating the plastic culture dishes with either collagen or fibrin clots. The formation of tight junctions between endothelial cells can be confirmed by electron microscopy. The tube formation assay is a viable assay, which can be accomplished within a day. Image analysis is used generally to measure the endothelial cells ability to form tube-like structures.
Further endothelial cell adhesion, migration and invasion have been reported [ 70 ]. Though very reliable in studying the reorganization, the TFA represents only a small part of angiogenesis and allows specific predictions of the nature of different endothelial cell strains rather than testifying the angiogenic process itself. There is a large variety of assays to measure cell proliferation.
Well-established ones are for example the MTT-assay, in which living cells reduce 3- 4,5-Dimethylthiazolyl -2,5-diphenyltetrazolium bromide, a yellow tetrazole to purple formazan, and the thymidine incorporation assay using BrdU 5-bromo-2'-deoxyuridine , an analogue of thymidine. Both assays are performed to detect proliferating cells in living tissue, Especially the BrdU incorporation assay is most frequently used for cell proliferation studies in angiogenesis.
The cells used in proliferation assays are endothelial cells. However, it matters from which source the endothelial cells are isolated since they differ not only between large-vessel-derived endothelial cells and ones originated from microvasculature. They also seem to have distinctive characteristics when obtained from different organs and even when taken from different sites in one single organ 71, 72, Another fact is the total difference between species, which cannot be ignored: One example are the pig and murine endothelial cells, which bind BSL-1 and BSL-4 lectin from Bandeira simplifolica and are therefore a target for hyper-immune destruction when implanted into patients.
The most common cell types used in this assay are either bovine aortic endothelial cells or human umbilical vein endothelial cells. During culture in the laboratory these cells are by nature in a proliferative state, while they are more in a quiescent, non-proliferative state in vivo. Therefore endothelial cells in vitro depend on the environment in which they are kept during culturing. Cell migration is the movement of cells from one area to another induced by chemical signals. Generally cell migration plays a vital role in processes like cell differentiation, tumor metastasis and wound healing.
In angiogenesis studies the focus is mainly laid on migrating endothelial cells.
In terms of angiogenesis one should speak of cell invasion, for endothelial primarily degrade the basal lamina before migrating toward an angiogenic stimulus. The assays most frequently performed are the blind-well chemotaxis chamber and the scratch-wound assay. The blind-well chemotaxis chamber is a modified Boyden chamber as used for classic neutrophil chemotaxis. Instead, endothelial cells are placed on a cell permeable filter.
When an angiogenic stimulus is added into the medium below the filter, the cells start to migrate. The system is very useful in concentration-dependent effects. In the other preferred assay, the scratch-wound assay [ 74 , 75 ], HUVECs are used to be seeded into trans-wells. The test is used to measure the time needed under different conditions drug treatment, etc. The basis of this assay is the excised aorta of rats or mice from which the connective tissue has been removed.
The aortic tube will then be cut into rings or small pieces aortic explants and taken into culture with collagen gel. Since angiogenesis in vivo not only involves endothelial cells, but also pericytes, the basal lamina and other surrounding cells, researchers have moved to asses angiogenesis by organ culture methods. Of these, the rat aortic ring assay has become one of the most widely used ones. It is even possible to recapitulate all of the key steps of endothelial cell sprouting or angiogenesis: Matrix degradation, that is to say breaking through the basal lamina, which is the second layer of mature vessel enveloping the endothelial cells, migration toward a signal source, proliferation and finally reorganization or tube-forming can be analyzed.
After several days in culture image analysis is used to measure length and abundance of the sprouting endothelial cells.
Breast tumour angiogenesis | Breast Cancer Research | Full Text
Aortic ring assay experiments. Phase-contrast images showing endothelial sprouting from aortic rings embedded in Matrigel and the method of quantification. To make the data comparable, we developed a tangents measure methods. Although the situation in the aortic ring assay is closer to the in vivo environment than in dissociated single cell culture assays, one has to deal with the higher experimental variability due to animal strains and animal age. The main factors here are the incomplete removal of the surrounding connective tissue which can affect the sprouting and outgrowth of endothelial cells, the tissue from multiple animals itself which can result in an inconstant angiogenic response and last, the evaluation of the images, for the sprouting is a three-dimensional process.
The aortic ring assay is a reliable assay to test substances for their potential to positively or negatively affect angiogenesis and is able to monitor angiogenesis activity. In Sandison et al.
Tumor Vasculature: The Achilles` Heel of Cancer ?
They implanted a transparent window chamber in the ear of a rabbit [ 77 ]. The preparation allows inspection of an in vivo environment from outside the body, a long-term monitoring of angiogenesis is possible.
Clark et al. Ide et al. Their results led to the proposal of a 'vessel growth- stimulating substance' produced by the tumor. The dorsal skinfold chamber is an adaption of the rabbit ear chamber made by Algire et al. A metal chamber with a transparent window is implanted in various animal species, in rodents it is the most widely used window preparation. The dorsal window can last days.
To study the effect of tumors on vessel formation, fluorescent tumor cells are implanted into the chamber and the angiogenic activity, infiltration by immune cells, tumor cell migration and tumor growth are then monitored by intravital microscopy IVM [ 83 , 84 ]. The main limitation of this model is the non-organotypical environment of tumors other than skin cancer.
Breast tumour angiogenesis
A widely performed assay to test angiogenesis in vivo is the chorioallantoic membrane CAM- assay. Initially, Murphy et al. Highly promoted by Folkman et al. Since the introduction of the CAM-assay almost a century ago multiple modifications of the original angiogenic assay were made which allow quantification of the angiogenic process. In addition chick embryos are immune-incompetent until embryonic day Therefore grafting of cells of different species e. These three reactions of the CAM are observed on the ninth day of embryonation. Test substances are applied directly onto the CAM and the membrane is scanned after five minutes for the above named reactions.
Further the test allows analysis for angiogenesis of tumor-models growing on the CAM. It was developed by Leighton et al. The chorioallantoic membrane vascular assay is considered suitable as an option to replace animal trials to study vascular effects. The preparation of the test starts with an egg four days after fertilization in which a small window over the CAM is cut.