Angiogenic factors and their function

1.8.2.1 Vascular endothelial growth factor activities Mitogenesis, angiogenesis, and endothelial survival

VEGF promotion of vascular endothelial cell (EC) growth derived from arteries, veins, and lymphatics has been well documented in-vitro (78). The vascular progenitors differentiate to ECs in response to VEGF. While A is a key regulator of blood vessel growth, VEGF-C and VEGF-D regulate lymphatic angiogenesis (89). Although endothelial cells are the primary targets of VEGF, several studies have reported mitogenic effects also on certain non-endothelial cell types, such as retinal pigment epithelial cells (90), pancreatic duct cells (85), and Schwann cells (86). VEGF has also been shown to stimulate surfactant production by alveolar type II cells, resulting in a protective effect from respiratory distress syndrome in mice (91).VEGF is a survival factor for endothelial cells, both in vitro and in vivo (92-94). In vitro, VEGF prevents endothelial apoptosis induced by serum starvation. Such activity is mediated by the phosphatidylinositol 3-kinase (PI3 kinase)/Akt pathway (93, 94).

Effects of VEGF on bone marrow cells and hematopoiesis

The earliest evidence of VEGF activity on blood cells was a report describing its ability to promote monocyte chemotaxis (95). Associations between monocyte function and angiogenic factors have been detailed further in Section 1.12.3. Subsequently, further effects of VEGF on hematopoietic cells have been described, including inducing colony formation by mature subsets of granulocyte-macrophage progenitor cells (79), increase production of B cells and the generation of immature myeloid cells (80). VEGF delivery to adult mice inhibits dendritic cell development (81), leading to the hypothesis that VEGF facilitates tumor growth by allowing escape of tumors from the host immune system (57).

Figure 1.2 VEGF and the VEGF-receptor system.

VEGF-A, a major contributor to angiogenesis, binds and activates VEGFR-1 as well as VEGFR-2, and regulates vasculogenesis, angiogenesis, inflammatory responses, and carcinogenesis. The soluble form of VEGFR-1 appears to be an important modulator for the placental vasculature. (With permission from Shibuya 2006 (96)).

Enhancement of vascular permeability and hemodynamic effects

The ability of VEGF to induce microvascular permeability is a step necessary for angiogenesis, by providing extravasation of fibrin, which represents a scaffold for endothelial cell proliferation and migration (97). This function may play an important role in inflammation and other pathological circumstances (80).

Several studies have pointed to the critical role of nitric oxide (NO) in VEGF-induced vascular permeability, as well as angiogenesis (98-100).

1.8.2.2 Role of VEGF in Physiological Angiogenesis

VEGF has been identified as a major determinant of angiogenesis in normal physiological processes.

VEGF A has been shown to have an essential role in vasculogenesis and angiogenesis in the mouse embryonic and postnatal development. VEGF C regulates lymphatic development (80). Inactivation of a single or both alleles of VEGF –A and C result in embryo lethality by day 12. VEGF is required not only for proliferation but also for survival of endothelial cells (101). VEGF gradient is needed for directional growth and cartilage invasion by metaphyseal blood vessels (102, 103). VEGF-dependent blood vessel recruitment is essential not only for coupling cartilage resorption with bone formation, but also for bone homeostasis (104).

VEGF plays an important role in reproductive homeostasis, being involved in the cyclic proliferation and regression of blood vessels in the endometrium, during ovarian follicle development (105, 106), oocyte fertilization and development and homeostasis of germ cells (9). VEGF has been established as the principal regulator of ovarian angiogenesis and plays a key role in corpus luteal development and function by establishing vasculature for delivery of cholesterol to luteal cells for progesterone biosynthesis (105, 107). Recently, VEGF-induced capillary network has been identified as essential for pancreatic cell angiogenesis and fine tuning blood glucose regulation (108).

VEGF and KDR expression has been found on type II pneumocytes, and VEGF directly stimulated surfactant production of type II pneumocytes in vitro (9, 109). These data indicate an important role for VEGF in fetal lung development via its effect on epithelial maturation,

independent of its pro-angiogenic effects. Genetic studies in mice have revealed an important role for VEGF and PlGF in renal development and disease. During glomerular development, podocytes express VEGF, Flt-1 and KDR (110). Conditional deletion of VEGF in glomerular podocytes resulted in nephron malformation and nephrotic syndrome (111).

1.8.2.3 Role of VEGF in Pathological Conditions Solid tumors and hematological malignancies

In situ hybridization studies have shown that VEGF mRNA is upregulated in many human

tumours, including carcinoma of the lung, breast, gastrointestinal tract, kidney, ovary, endometrium and glioblastoma multiforme (80). Clinical trials in cancer patients are ongoing with several VEGF inhibitors, including a humanized anti-VEGF monoclonal antibody and an anti-KDR antibody bevacizumab (Avastin) (80). Thrombosis, increased blood pressure, and proteinuria were among the side effects of treatment in initial studies (112).

Intraocular neovascular syndromes

Neovascularization and vascular leakage are major cause of visual loss in diabetes mellitus, occlusion of central retinal vein, oxygen exposure in prematurity as well as age-related macular degeneration (113, 114). Animal studies have clearly shown the role of VEGF as a mediator of ischaemia induced intraocular neovascularization (115).

Inflammation and brain edema.

VEGF up-regulation has been implicated in various inflammatory disorders such as psoriasis, rheumatoid arthritis, brain edema as well as in keratinocytes in wound healing (80).Vascular endothelial growth factor has been shown to have anti-apoptotic activity, and whether it plays a role in abnormal placental apoptosis is unknown.

1.8.2.4 Placental growth factor activities

Although the exact physiological actions of PlGF are not clear, evidence suggests a pivotal role for PlGF in regulating VEGF-dependent angiogenesis under pathological conditions (85).

PlGF has been hypothesized to play a role in placental development and angiogenesis. PlGF is a very weak stimulator of endothelial chemotaxis and proliferation, and when binding competition studies are performed with extracellular domains from either Flk-1/KDR or Flt-1, PlGF appears to be able to bind to Flt-1 but not to Flk-1/KDR (116). PlGF homodimers bind Flt-1 and NRP-1 while PlGF/VEGF-A heterodimers bind KDR and Flt-1/KDR heterodimers in vitro (24).

Evidence to date suggests that, whereas the VEGF/KDR axis is important for physiological angiogenesis and pathological angiogenesis, the PlGF/Flt axis appears to be specifically crucial for pathological angiogenesis by modulating the effects of the VEGF/KDR axis (9).

Proposed mechanisms by which PlGF potentiates angiogenesis include stimulating endothelial cells via Flt-1, separating VEGF-A from Flt-1 by competing for the receptor, allowing VEGF-A to activate KDR, recruiting monocytes/macrophages which have a crucial role in vessel growth (88) and inducing the secretion of VEGF-A from monocytes (117).

Monocytes/macrophages have been proposed as critical players in the process of angiogenesis and wound healing. The strong placental expression of PlGF could contribute to the increased demand for angiogenesis in the growing placenta which may be partially mediated by chemo-attraction of peripheral blood monocytes (9).