PriCells: Primary cells culture with supplement of vascular endothelial growth factor (VEGF)

Introduction
Vascular endothelial growth factor (VEGF) is a signal protein produced by cells that inspires the growth of new blood vessels.  It is part of the system that re-establishes the oxygen supply to tissues when blood circulation is inadequate.  The function of VEGF is to generate new blood vessels during embryonic development, new blood vessels after injury, muscle following exercise, and new vessels (collateral circulation) to bypass blocked vessels. When VEGF is over-expressed, it can contribute to disease. Solid cancers cannot grow beyond a limited size without an adequate blood supply; cancers that can express VEGF are able to grow and metastasize.  Over-expression of VEGF can cause vascular disease in the retina of the eye and other parts of the body. Drugs such as bevacizumab can inhibit VEGF and control or slow those diseases.  VEGF is a sub-family of growth factors, to be specific, the platelet-derived growth factor family of cystine-knot growth factors.  They are important signaling proteins involved in both vasculogenesis (the de novo formation of the embryonic circulatory system) and angiogenesis (the growth of blood vessels from pre-existing vasculature).

Classification
The broad term 'VEGF' covers a number of proteins from two families, that result from alternate splicing of mRNA from a single, 8-exon, VEGF gene.  The two different families are referred to according to their terminal exon (exon 8) splice site - the proximal splice site (denoted VEGFxxx) or distal splice site (VEGFxxxb).  In addition, alternate splicing of exon 6 and 7 alters their heparin-binding affinity, and amino acid number (in humans: VEGF121, VEGF121b, VEGF145, VEGF165, VEGF165b, VEGF189, VEGF206; the rodent orthologs of these proteins contain one fewer amino acid).  These domains have important functional consequences for the VEGF splice variants, as the terminal (exon 8) splice site determines whether the proteins are pro-angiogenic (proximal splice site, expressed during angiogenesis) or anti-angiogenic (distal splice site, expressed in normal tissues).  In addition, inclusion or exclusion of exons 6 and 7 mediate interactions with heparan sulfate proteoglycans (HSPGs) and neuropilin co-receptors on the cell surface, enhancing their ability to bind and activate the VEGF receptors (VEGFRs).

Biology
All members of the VEGF family stimulate cellular responses by binding to tyrosine kinase receptors (the VEGFRs) on the cell surface, causing them to dimerize and become activated through transphosphorylation, although to different sites, times and extents.  The VEGF receptors have an extracellular portion consisting of 7 immunoglobulin-like domains, a single transmembrane spanning region, and an intracellular portion containing a split tyrosine-kinase domain. VEGF-A binds to VEGFR-1 (Flt-1) and VEGFR-2 (KDR/Flk-1).  VEGFR-2 appears to mediate almost all of the known cellular responses to VEGF.  The function of VEGFR-1 is less well-defined, although it is thought to modulate VEGFR-2 signaling.  Another function of VEGFR-1 may be to act as a dummy/decoy receptor, sequestering VEGF from VEGFR-2 binding (this appears to be particularly important during vasculogenesis in the embryo).  VEGF-C and VEGF-D, but not VEGF-A, are ligands for a third receptor (VEGFR-3), which mediates lymphangiogenesis.

Medicine
VEGFxxx has been implicated with poor prognosis in breast cancer. Numerous studies show a decreased overall survival and disease-free survival in those tumors overexpressing VEGF.  The overexpression of VEGFxxx may be an early step in the process of metastasis, a step that is involved in the "angiogenic" switch.  Although VEGFxxx has been correlated with poor survival, its exact mechanism of action in the progression of tumors remains unclear.  VEGFxxx is also released in rheumatoid arthritis in response to TNF-α, increasing endothelial permeability and swelling and also stimulating angiogenesis (formation of capillaries).  VEGFxxx is also important in diabetic retinopathy (DR).  The microcirculatory problems in the retina of people with diabetes can cause retinal ischaemia, which results in the release of VEGFxxx, and a switch in the balance of pro-angiogenic VEGFxxx isoforms over the normally expressed VEGFxxxb isoforms.  VEGFxxx may then cause the creation of new blood vessels in the retina and elsewhere in the eye, heralding changes that may threaten the sight.  VEGFxxx plays a role in the disease pathology of the wet form age-related macular degeneration (AMD), which is the leading cause of blindness for the elderly of the industrialized world.  The vascular pathology of AMD shares certain similarities with diabetic retinopathy, although the cause of disease and the typical source of neovascularization differes between the two diseases.  VEGF-D serum levels are significantly elevated in patients with angiosarcoma.  Once released, VEGFxxx may elicit several responses. It may cause a cell to survive, move, or further differentiate.  Hence, VEGF is a potential target for the treatment of cancer.  The first anti-VEGF drug, a monoclonal antibody named bevacizumab, was approved in 2004.  Approximately 10-15% of patients benefit from bevacizumab therapy; however, biomarkers for bevacizumab efficacy are not yet known. VEGFs are not the only promoters of angiogenesis.  In particular FGF2 and HGF are potent angiogenic factors.  Patients suffering from pulmonary emphysema have been found to have decreased levels of VEGF in the pulmonary arteries.  In the kidney, increased expression of VEGFxxx in glomeruli directly causes the glomerular hypertrophy that is associated with proteinuria.