Sprouting of endothelial cells is an early event in angiogenesis, which follows vasodilation and degra- dation of matrix (Kurz, 2000) and it represents a valuable target for therapies because it takes place so early in the angiogenic process. The degradation of matrix is accomplished by the family of matrix
metalloproteinases (MMPs) (Caserman and Lah, 2004). The mechanisms by which sprouts progress to form a lumen and ultimately become competent to support blood flow are largely unknown. Therefore, the study of the early steps of vessel sprouting can point to new therapeutic directions once key targets in these pathways have been identified.
The most promising in-vitro assays for eluci- dating relevant molecules and pathways necessary for endothelial cell morphogenesis are those using three-dimensional extracellular matrices, because endothelial cells experience a richer, more complex physical environment than cells cultured on two- dimensional surfaces (Ucuzian and Greisler, 2007). The collagen I and fibrin matrices represent the major matrix environments where angiogenic events
Organ Disease manifestation Comments
Blood vessels Vascular malformations caused by abnormal Localised lesions due to vascular malformation; congestive remodelling, haemangioma and atherosclerosis heart failure resulting from atherosclerosis
from increased vascularisation
Eye Diabetic retinopathy and wet age-related macular Blindness due to leaky vessels in diabetic retina; blindness degeneration from increased vascularisation from proliferating choroidal blood vessels
Skin Psoriasis from increased vascularisation that Psoriasis appears as scaly, raised red lesions as a common becomes tortuous and enlarged; decubitus (stasis) form of this disease; stasis ulcers are open surface wounds ulcers from insufficient vascularisation; Kaposi’s that fail to heal
sarcoma, allergic oedema, and neoplasms from increased vascularisation
Bone and joints Increased vascularisation of synovial joints in Inflammation of synovium in rheumatoid arthritis leads to arthritis and of bone tissue in cancers joint destruction; destruction of cartilage in osteoarthritis
causes pain and impaired mobility
Heart, skeletal muscle Increased vascularisation of heart due to work Contractile dysfunction of heart tissue leads to heart failure; overload; ischaemic heart and limb disease from coronary heart disease manifests as a result of occlusion of insufficient vascularisation blood vessels and poor oxygen supply
Adipose tissue Increased vascularisation of fat tissue Fat cells accumulate around new blood vessels causing obesity
Uterus, ovary Increased vascularisation of uterine tissue, Uterine tissue becomes dysfunctional from excessive endometrium, ovary bleeding; endometriosis can cause ectopic pregnancy,
miscarriage and also infertility
Brain Increased vascularisation in brain tumours; Gliomas and glioblastomas are incurable diseases of brain; insufficient vascularisation of brain can lead to stroke can incapacitate the cognitive and functional aspects
strokes of the brain
Table 13.1 Angiogenesis in human diseases
take place (Ryan and Barnhill, 1983). For example, during endothelial sprouting there is the induced expression of endogenous growth factors, transcrip- tion factors and signalling molecules, endothelial cell differentiation markers and adhesion molecules and a marked down-regulation of positive regulators of the cell cycle and ubiquitin-proteasome genes (Bell et al., 2001). In stark comparison, the angiogenesis- screening assay using the basement-membrane matrix Matrigel, which measures the ability of endothelial cells to form a meshwork of cords on a tumour cell-derived matrix is markedly independent of transcriptional events (Zimrin et al., 1995), and protein synthesis (Laterra and Goldstein, 1991). These and other drawbacks with the Matrigel gel assay (Vernon et al., 1992) limit its scope for screening purposes. In the endothelial cell sprouting
assay (3D-ECSA) (Korff and Augustin, 1998), endothelial cells are induced over a period of 24 h to form spheroids by aggregating. The spheroids are next seeded in suspension in a collagen I matrix by gelling at 37⬚C. Exogenous growth factors, such as VEGF, when added to the three-dimenional culture, stimulate the growth of vessel-like structures that grow out from the spheroid. Extracts and drugs being tested for angiogenesis inhibition are added along with VEGF. The sprouting extent and its inhi- bition are observed after a period of 18–24 h, which allows one to readily identify agents that block vessel development. The assay has been used by our labo- ratory to identify several classes of angiogenesis inhibitors, one of which is withaferin A from the medicinal plant Withania somnifera (Mohan et al., 2004).
Assay Measurement Comments
Cell proliferation Inhibition of cell doubling opposing stimulatory Cytostatic activity blocks cell proliferation without causing effect of a defined angiogenic factor cell death
Cell migration Inhibition of cell migration opposing stimulatory The extension of endothelial cell processes allows cells effect of a defined angiogenic factor such as VEGF to migrate over a substratum
or bFGF
Invasion Inhibition of cell invasion opposing stimulatory The growth of endothelial cells through a porous effect of a defined angiogenic factor membrane or matrix in response to a chemotactic factor Sprouting Inhibition of migration, invasion and tube formation An integrated assay which couples vascular invasion, tube
in a 3D matrix of collagen I or fibrin opposing formation and maturation in 3D matrix stimulatory effect of a defined angiogenic factor
Matrigel cord assay Inhibition of cord assembly by endothelial cells on Endothelial cells assemble into cords over the matrix complex matrix derived from tumour stroma
opposing stimulatory effect of a defined angiogenic factor
CAM in vivo Inhibition of blood vessel growth in the CAM of a The developing vasculature of the CAM is highly sensitive fertilised developing chicken egg inhibitors of angiogenesis
Corneal angiogenesis Inhibition of de novo capillary growth in cornea Blood vessels from surrounding scleral vessel supply invade in vivo opposing stimulatory effect of a defined angiogenic the avascular cornea in response to slow-release growth
factor factor implanted in cornea
Matrigel in vivo Inhibition of blood vessel growth into a Matrigel Blood vessels invade the Matrigel plug in response to plug implanted in abdominal region of mouse stimulus from growth factor impregnated plug
Table 13.2 In vitro and in in vivo angiogenesis assays