The formation of blood vessels is one of the major events in organ development and repair. Examples of normal blood vessel formation are found in the complex developing network of arteries, veins and capillaries in growing fetuses or infants, during tissue regeneration, wound healing, and also in the cyclical menstrual changes of the uterine lining in mature females. Pathological processes in blood vessels occur in vascular diseases, cancer and metastasis. Cancer cells that penetrate the walls of blood vessels are able to circulate in the bloodstream, spread and grow in normal tissues. A fundamental issue in cancer cell biology involves deciphering and understanding of the processes that allow cancer cells to metastasize, migrate and form tumors in other tissues.

The development of new blood vessels is believed to be an essential event favoring cancer cell growth. During tumor angiogenesis, new networks of blood vessels are created that supply the essential nutrients and oxygen needed by the growing tumors. Endothelial cells, lining the lumina of blood vessels, divide on the basis of signals received. The processes regulating endothelial cell growth involve two antagonistic varieties of regulatory proteins: activators and inhibitors of angiogenesis.

Vascular endothelial growth factor (VEGF) acts as a critical mediator of tumor angiogenesis. Pharmacological manipulation of VEGF expression is therefore of considerable interest in current attempts to inhibit or retard tumor growth. Angiogenesis functions as a signaling cascade. Initially, VEGF is synthesized in the tumor cells, secreted to the surrounding tissue, and binds to specific VEGF receptors present on the surface membrane of endothelial cells. This specific VEGF binding activates several other proteins resulting in the activation of genes in the nucleus of endothelial cells that stimulate endothelial cell growth.

VEGF is an endothelial-cell specific mitogen and a potent angiogenic factor. VEGF has been demonstrated, both in vitro and in vivo, to be a critical mediator of angiogenesis in various disease processes including: diabetic retinopathy, tumor angiogenesis and coronary artery disease. Pharmacological manipulation of VEGF in these disorders, either to inhibit or to augment angiogenesis, requires an understanding of the molecular mechanisms regulating VEGF.

The VEGF locus spans 14 kb with eight exons. Alternative splicing results in the generation of mRNAs encoding for at least five different isoforms of 121, 145, 165, 189 and 206 amino acids. The mRNA encoding all these isoforms contains a 1 kb 5? untranslated region (UTR) and a 1.8 kb 3? UTR. The VEGF mRNA 5? UTR is unusual in having a short upstream open reading frame and in being enriched in guanine–cytosine (GC) residues. The VEGF 3? UTR contains four canonical polyadenylation sites, although only the 5? and 3? sites are used.

All VEGF isoforms contain a classic signal peptide sequence allowing its efficient secretion from the cell, but the fate and bioavailability of the VEGF protein is isoform specific. For example, VEGF 121 and 165 amino acid isoforms are predominantly secreted, whereas the 189 and 206 amino acid isoforms are almost exclusively cell-associated, presumably via association with cell surface proteoglycans such as heparan sulfate. The VEGF protein acts in a paracrine manner after secretion from the cell binding to specific surface membrane-bound receptors on endothelial cells. This initiates a signal transduction cascade leading to endothelial cell proliferation and angiogenesis. An intracrine role for VEGF has not yet been described.

Regulation of expression of the VEGF gene is extremely important. Hypoxia is a physiological condition that, in addition to hormones, interleukins, insulin, and growth factors, induces angiogenesis. During hypoxia, three different levels of regulation ensure increased VEGF expression. Firstly, hypoxia-inducing factor 1 (HIF1) is a transcription factor that mediates increased transcription of VEGF mRNA during hypoxia. Secondly, HuR acts as a RNA-binding protein that binds exclusively to a region in the 3? UTR of the mRNA and mediates increased mRNA stability with hypoxia. VEGF mRNA stability appears to be of primary importance, not only for the induction of VEGF in response to physiological stimuli, but also for the constitutively elevated levels of VEGF observed in a wide variety of tumors. At the translation stage, there are internal ribosome entry sites (IRES) at the 5? UTR region of VEGF mRNA that maintain the translational efficiency of VEGF under hypoxic conditions during which cap-dependent translation is inhibited.

t is a widely accepted dogma that VEGF acts in a paracrine fashion after binding to specific receptors on endothelial cells leading to stimulation of endothelial cell growth and angiogenesis. However, it also appears that VEGF has an intracrine function. Nuclear VEGF is a challenging new concept that may help to understand other functions played by this multi-potent molecule, including regulation of its own expression.

HuR protein is a member of the embryonic lethal, abnormal vision, Drosophila-like (ELAVL) protein family. It has a sequence length of 1230 bases. ELAVL1 contains 3? RNA-binding domains and binds cis-acting adenine–uracil (AU)-rich elements. It destabilizes mRNAs and thereby regulates gene expression. We were able to show a specific increase of HuR mRNA degradation using methods that knockout stabilization sequences. The RNA interference (RNAi) assay is a post-transcriptional gene silencing (PTGS) induced by the direct introduction of double-stranded RNA (dsRNA) in animal cells. In this process, the introduction of dsRNA in a cell inhibits gene expression in a sequence-dependent fashion. The mediators of RNAi are 21- and 22-nucleotide small interfering RNAs (siRNA). The siRNAs bind to a ribonuclease complex called RNA-induced silencing complex (RISC) that guides the small dsRNAs to its homologous mRNA target. Consequently, RISC cuts the mRNA approximately in the middle of the region paired with the antisense siRNA, after which the mRNA is further degraded.

Using a chemically synthesized siRNA designed to target specifically HuR mRNA to abolish HuR expression, a high level of gene silencing was obtained following transfection in either COS or 293 cells, with different amounts of siRNA. After transfection, cells were harvested and total cell protein preparations were obtained with lysis buffer. The same amount of protein was loaded on a 10% SDS–polyacrylamide gel electrophoresis and subjected to immunoblot analysis. HuR was identified using a monoclonal anti-HuR antibody and was detected with the ECL system. In parallel, an indirect immunofluorescence experiment was performed in which a monoclonal anti-HuR antibody was the first antibody and a rhodamine-conjugated anti-mouse IgG antibody was the secondary antibody. Cells were observed under a fluorescence microscope or confocal microscope. siRNA designed to degrade annexin (endogenous anti-inflammatory protein/mediator of glucocorticoid actions in inflammation) was used as control siRNA to show specificity of siRNA against HuR.

Cell fractionation was performed in cells grown under conditions of both normoxia and hypoxia. In a Western blot, HuR expression during hypoxia shows an increase in the nuclear fraction as compared to the cytoplasmic fractions. In normoxia, the pattern is the same in both cytoplasm and nucleus. As a control, the eukaryotic initiator factor 4A (eIF-4A), a RNA helicase factor, that is, essentially cytoplasmic and melts the 5? secondary structure of the mRNA, was used. It is a 50 kDa polypeptide and was shown, as expected, to be cytoplasmic under both normoxia and hypoxia conditions.