The formation of new blood vessels in the body are generated by three processes: vasculogenesis, arteriogenesis and angiogenesis.
Angiogenesis is a tightly regulated process that is balanced by pro- and antiangiogenic signals including integrins, chemokines, angiopoietins, oxygen sensing agents, junctional molecules and endogenous inhibitors.4 It is a hallmark of over 50 different disease states, and its dysfunction is implicated in cancer, psoriasis, various eye diseases, rheumatoid arthritis, asthma and other autoimmune diseases, infectious diseases, coronary arterial diseases, stroke, atherosclerosis and impaired wound healing, among others.1, 5
Figure 1. Mechanisms of Angiogenesis(A) Angiogenic stimuli give rise to a cytokine gradient and activate endothelial cells. (B) Endothelial cells break down the basement membrane, sprout toward the external gradient and begin to migrate toward the angiogenic stimulus. Pericytes detach from the blood vessel. (C) Reassembly of endothelial cells and formation of new cell-cell contacts and vessel lumina. (D) Vessel stabilization by pericyte recruitment and initiation of blood flow.
The cell culture tube formation assay first described in 1988 by Kubota et al.7 is one of the most widely used in vitro assays for angiogenesis. The assay involves plating endothelial cells onto a basement-membrane-like substrate on which the cells form tubules within six to 20 hours. These tubules mostly contain a lumen, and the cells develop tight cell-cell and cell matrix contacts. Quantification can be performed by measuring the tube area or the length and/or number of branch points, with area measurement being the most common and easiest method.6; 10 This powerful semiquantitative assay has many advantages. It is quick and easy to perform, can be scaled up for high-throughput analysis, and factors can be added exogenously to the medium, transfected into the cells, or knocked down.9
The basement membrane is an important extracellular matrix found in all endothelial tissues. It maintains tissue integrity, serves as a barrier between cells and proteins, separates different tissue types, transduces mechanical signals, has many biological functions that help maintain tissue specificity, and acts as a storage depot for growth factors and enzymes. Its major components are laminins, collagen IV, heparan sulfate proteoglycans, various growth factors, cytokines, chemokines and proteases. For the tube formation assay, basement membrane can be prepared from tissues or tumors, 11 or else a gel matrix layer of fibrin, collagen or Matrigel/ECM Gel can be used. The type of matrix used is important, as different matrices result in different rates of differentiation. It appears that the mechanisms by which endothelial cells differentiate to form tubes are at least partly dependent upon the matrix onto which the cells are plated; it is therefore advisable to test substances on more than one matrix type.6
A crucial aspect for ensuring successful experiments is the right choice of endothelial cells. The tube-forming capacity varies among different groups of endothelial cells, making it essential to choose the assay conditions and cell types that most closely resemble the angiogenic conditions or disease being studied. In adult humans, there is a high degreeof heterogeneity among endothelial cells along the vascular tree. This facilitates biological adaptation to local requirements. Functional heterogeneity among endothelial cells is involved in controlling vasoconstriction and vasodilation, blood coagulation, fibrinolysis, antigen presentation, atherogenesis, and catabolism of lipoproteins. There is also considerable heterogeneity in endothelial cells derived from different locations within the body, related to the microenvironmental conditions in each organ and the specialized roles that the endothelium plays in them.6
PromoCell supplies a wide variety of endothelial cells from dermal, cardiac, pulmonary, uterine and saphenous tissuesas well as from large vessels and umbilical cords. Theoretically, all these endothelial cells ought to be able to build tubes under the right conditions. However, the extent of tube formation and their responses to angiogenesis stimulators and inhibitors vary greatly depending on the cell type. The tube formation assay presented in the following section has been tested for HUVEC, HDMEC, HCAEC, HPMEC, HAoEC and HCMEC in particular.
The tubular network in the wells can be imaged without fixation or labelling using an inverted microscope ( Fig. 2A and C).
Prepare 6 μM of Calcein AM solution (17783) in recommended basal medium. Add 50 μL of the solution per well without aspirating the medium. Incubate the plate in a humidified incubator (37 °C, 5% CO2) for 30 min. Calcein AM-labelled cells can be observed immediately using an inverted fluorescence microscope with 485 nm excitation or 520 nm emission filter.
Prepare 0.1% Crystal Violet staining solution (V5265) in distilled water (w/v). Carefully remove the medium from the wells and wash the cells with 100 μL PBS per well. Remove the water and add 100 μL of Crystal Violet solution per well. Incubate the plate for 15–30 min at room temperature. Wash the cells twice using sterile water and image cells using an inverted microscope (Fig. 2D).
Figure 2.In vitro endothelial cell tube formation assay. (A) Light microscopy image of PromoCell HUVECs cultured on Basement Membrane Extract for 17 hours. (B) Calcein staining of PromoCell HUVECs cultured on Basement Membrane Extract for 17 hours using PromoKine’s Angiogenesis Assay Kit. (C) Light microscopy image of PromoCell HCAECs cultured on Basement Membrane Extract and stimulated with 50 ng/ml FGF for 16 hours. (D) Crystal Violet staining of PromoCell HCAECs cultured on BME for 16 hours.
To continue reading please sign in or create an account.Don't Have An Account?