There are a number of factors that have been known to induce angiogenesis. Among the known angiogenesis factors, vascular endothelial growth factor (VEGF) serves as a major regulator of the angiogenic processes and has been the center of numerous investigations. The VEGF family of glycoproteins is composed of VEGF-A, VEGF-B, VEGF-C, VEGF-D, and placenta growth factor (PIGF). VEGF-A, also known as vascular permeability factor (VPF), has four main isoforms (VEGF121, VEGF165, VEGF189, and VEGF206) produced by alternative splicing. A fifth isoform VEGF145 is found within the reproductive tract of females, and has also been found in placenta and carcinoma cells. VEGF189 and VEGF206 are the most abundant forms. VEGF189 is present throughout the tissues, whereas VEGF206 is only found in embryonic tissue. Both are sequestered in the extracellular matrix. VEGF165 and VEGF121 are the most frequently secreted, however, VEGF165 is the mature isoform.
VEGF stimulates migration and proliferation of microvascular endothelial cells derived from arteries, veins, and lymphatic vessels as well as angiogenesis in vivo and in vitro. For example, Phillips, et al. reported that VEGF stimulated direct angiogenesis in the rabbit cornea. Similarly, Tolentino, et al. reported that VEGF was sufficient to produce iris neovascularization and neovascular glaucoma in primates. VEGF also induces lymphangiogenic responses in mice. When serum is lacking, VEGF prevents apoptosis of endothelial cells in vitro. Cells of many human solid tumors express large amounts of VEGF protein, thus stimulating the development of new blood vessels within the developing tumor.
Hypoxia plays a major role in regulating the ex
During hypoxia, prolyl hydroxylation is inhibited, blocking VHL-mediated degradation, resulting in the accumulation of HIF-1α within the cell. This in turn activates ex
Interleukin-8 (IL-8), which is produced by macrophages, is another important mediator of tumor-derived angiogenesis. IL-8 is normally secreted in response to growth factors, inflammatory cytokines, and pathophysiologic conditions. The increased ex
IL-8 has been shown to induce proliferation and chemotaxis of human umbilical vein endothelial cells (HUVEC) and human aortic endothelial cells. IL-8 has also been shown to stimulate both endothelial proliferation and capillary tube formation in vitro; the effect can be reversed using neutralizing antibodies to IL-8.
Tumor Microenvironment and Angiogenesis
Angiogenesis is crucial for tumor development. Solid tumors cannot grow beyond a few millimeters without a sufficient blood supply. As stated previously, angiogenesis depends on a balance between angiogenic inhibitors and inducers. During tumor progression, this balance is tipped favoring proangiogenic events and the angiogenic switch is turned on.
The tumor microenvironment contains a mixture of extracellular matrix (ECM) proteins, tumor cells, endothelial cells, fibroblasts and immune cells that turn the angiogenic switch on. The extracellular matrix provides structural support to cells and tissues, transmits signals through receptors, and binds and stores growth factors and other active molecules. Basement membranes (BM) are ECM that function as barriers, polarize cells, shape tissue structures and support migrating cells. Vascular BM support blood vessel endothelial cells and modulate endothelial cell behavior.
Many ECM proteins, such as collagens and laminins, have proangiogenic properties. They help promote endothelial cell survival, proliferation and migration. Many pro-angiogenic factors such as VEGF, basic fibroblast growth factor (bFGF) and transforming factor beta (TGF-β) are sequestered in the ECM and can be mobilized during ECM degradation by proteases secreted by tumor or stromal cells.
TAMs serve as the main source for chemokines, growth factors, cytokines and proteases that regulate endothelial cells in the tumor microenvironment. Studies using MCP-1-expressing tumor cells indicate that low levels of monocyte chemotactic protein-1 (MCP-1) have a modest effect on macrophage recruitment and enhanced angiogenesis and tumor growth in melanoma xenograft models. In contrast, elevated levels of MCP-1 induce more extensive macrophage infiltration, resulting in robust angiogenic responses, enhanced tumor growth and tumor regression.
Growth factors, including vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF), epidermal growth factor, and transforming growth factor-α (TGF-α), are known to be produced by TAMs. Among them, VEGF can promote endothelial cell growth, maturation and survival. VEGF functions as a monocyte chemotactic factor to attract TAMs to the hypoxic areas of tumor.
Inflammatory cytokines have been known to participate in macrophage-associated angiogenesis during wound healing. IL-8 is a mitogen of endothelial cells and stimulates angiogenesis. The conditioned media of macrophages has been shown to have similar effects and the effects can be abolished by neutralizing antibodies to IL-8, suggesting that IL-8 produced by macrophages plays a role in macrophage-associated angiogenesis. Tumor necrosis factor-α (TNF-α) is also involved in macrophage-associated angiogenesis. When overexpressed in endothelial cells, TNF-α was growth-inhibitory and cytotoxic to endothelial cells. In contrast, exogenous TNF-α promotes formation of new blood vessels in several in vivo models. Leibovich, et al. further showed that anti-TNF-α antibodies neutralized angiogenic activity in the conditioned media of activated macrophages, supporting the role of TNF-α as an angiogenic molecule from macrophages.