Shu-Ching Shih

Regulation of Human Vascular Endothelial Growth Factor mRNA Stability in Hypoxia by Heterogeneous Nuclear Ribonucleoprotein L

A 126-base region of human vascular endothelial growth factor (VEGF) 3′-untranslated region, which we identified as the hypoxia stability region, forms seven hypoxia-inducible RNA-protein complexes with apparent molecular masses ranging from 40 to 90 kDa in RNA-UV-cross-linking assays. In this study, we show that proteins that form the 60-kDa RNA-protein complex with the hypoxia stability region were present in both cytoplasmic and nuclear compartments. We purified the protein associated in the 60-kDa complex and identified it as heterogeneous nuclear ribonucleoprotein L (hnRNP L) by protein sequencing. Removal of hnRNP L by immunoprecipitation specifically abolished formation of the 60-kDa complex. Synthetic deoxyribonucleotide competition studies defined the RNA-binding site of hnRNP L as a 21-base-long sequence, 5′-CACCCACCCACAUACAUACAU-3′. Immunoprecipitation of hnRNP L followed by reverse transcription-polymerase chain reaction showed that hnRNP L specifically interacts with VEGF mRNA in hypoxic cells in vivo. Furthermore, when M21 cells transfected with antisense oligodeoxyribonucleotide to the hnRNP L RNA-binding site, the VEGF mRNA half-life was significantly reduced under hypoxic conditions. Thus, we propose that specific association of hnRNP L with VEGF mRNA under hypoxia may play an important role in hypoxia-induced post-transcriptional regulation of VEGF mRNA expression.

Nonvascular role for VEGF: VEGFR-1, 2 activity is critical for neural retinal development

The purpose of this study was to evaluate the function of extravascular vascular endothelial growth factor (VEGF) receptors in developing neural retina. VEGF is routinely described as a vascular endothelial cell‐specific mitogen, and VEGF receptor 1 (VEGFR‐1) and VEGF receptor 2 (VEGFR‐2) are described as endothelial cell specific, but there is evidence that these VEGF receptors are found outside the vasculature in neural tissue. The developing eye presents a unique opportunity to examine the function of VEGF in neural tissue alone. The peripheral retina is normally avascular at birth and becomes vascularized over the first 2 wk after birth. We localized VEGFR‐1 and ‐2 mRNA and protein to extravascular neuronal tissue during early retinal development. Avascular cornea also expresses these receptors. Inhibition of VEGFR‐1 and ‐2 in vivo with a specific small‐molecule tyrosine kinase antagonist, SU5416, inhibits development of the nonvascularized immature retina, resulting in cell loss in the inner retina, including the inner nuclear layer containing Muller cells and the ganglion cell layer containing astrocytes. Isolated retinal Muller cells express both VEGF receptors. VEGF stimulation activates MAPK, which is abrogated with inhibition of the receptors. We conclude that VEGFR‐ 1 and ‐2 are necessary for normal neural retinal development independent of vascular development.

Selective stimulation of VEGFR-1 prevents oxygen-induced retinal vascular degeneration in retinopathy of prematurity.

Oxygen administration to immature neonates suppresses VEGF-A expression in the retina, resulting in the catastrophic vessel loss that initiates retinopathy of prematurity. To investigate the mechanisms responsible for survival of blood vessels in the developing retina, we characterized two VEGF-A receptors, VEGF receptor-1 (VEGFR-1, also known as Flt-1) and VEGF receptor-2 (VEGFR-2, also known as Flk-1). Surprisingly, these two VEGF-A receptors differed markedly during normal retinal development in mice. At 5 days postpartum (P5), VEGFR-1 protein was colocalized with retinal vessels, whereas VEGFR-2 was detected only in the neural retina. Real-time RT-PCR identified a 60-fold induction of VEGFR-1 mRNA in retina from P3 (early vascularization) to P26 (fully vascularized), and no significant change in VEGFR-2 mRNA expression. Placental growth factor-1 (PlGF-1), which exclusively binds VEGFR-1, decreased hyperoxia-induced retinal vaso-obliteration from 22.2% to 5.1%, whereas VEGF-E, which exclusively binds VEGFR-2, had no effect on blood vessel survival. Importantly, under the same conditions, PlGF-1 did not increase vasoproliferation during (a). normal vessel growth, (b). revascularization following hyperoxia-induced ischemia, or (c). the vasoproliferative phase, indicating a selective function supporting blood vessel survival. We conclude that VEGFR-1 is critical in maintaining the vasculature of the neonatal retina, and that activation of VEGFR-1 by PlGF-1 is a selective strategy for preventing oxygen-induced retinal ischemia without provoking retinal neovascularization.

Molecular Profiling of Angiogenesis Markers

The goal of this study was to develop a sensitive, simple, and widely applicable assay to measure copy numbers of specific mRNAs using real-time quantitative reverse transcriptase-polymerase chain reaction (RT-PCR), and identify a profile of gene expression closely associated with angiogenesis. We measured a panel of nine potential angiogenesis markers from a mouse transgenic model of prostate adenocarcinoma (TRAMP) and a mouse skin model of vascular endothelial growth factor (VEGF)-driven angiogenesis. In both models, expression of VEGF correlated with expression of mRNAs encoding other angiogenic cytokines (angiopoietin-1 and angiopoietin-2), endothelial cell receptor tyrosine kinases (Flt-1, KDR, Tie-1), and endothelial cell adhesion molecules (VE-cadherin, PECAM-1). Relative to control, in dermis highly stimulated by VEGF, the Ang-2 mRNA transcript numbers increased 35-fold, PECAM-1 and VE-cadherin increased 10-fold, Tie-1 increased 8-fold, KDR and Flt-1 each increased 4-fold, and Ang-1 increased 2-fold. All transcript numbers were correspondingly reduced in skin with less VEGF expression, indicating a relationship of each of these seven markers with VEGF. Thus, this study identifies a highly efficient method for precise quantification of a panel of seven specific mRNAs that correlate with VEGF expression and VEGF-induced neovascularization, and it provides evidence that real-time quantitative RT-PCR offers a highly sensitive strategy for monitoring angiogenesis.

Fibroblast Growth Factor 2 Activation of Stromal Cell Vascular Endothelial Growth Factor Expression and Angiogenesis

Angiogenesis is a key component of human cancer progression and metastasis. In an effort to recapitulate early events in tumor-induced angiogenesis, we have employed a subcutaneous Matrigel implant model using immunodeficient mice as hosts. Matrigel-containing fibroblast growth factor 2 (FGF-2; 1.2 μg/ml) induced stromal cell infiltration into the Matrigel/skin interface within 4 days and maximal neovascularization at 7 days. Cells staining positive for the endothelial cell marker, platelet-endothelial cell adhesion molecule 1 (PECAM-1), were present in neovessels and in isolated cells within the Matrigel matrix. Immunohistochemical analysis revealed high levels of vascular endothelial growth factor (VEGF) deposited in the stromal interface present only in the FGF-2–containing but not in control Matrigel implants. VEGF expression was confirmed with in situ hybridization. High VEGF mRNA levels were observed in the infiltrating stromal cells but not in endothelial or endothelial precursors as defined by PECAM-1 staining. In vitro analysis of FGF-2–treated embryonic fibroblasts, Balb/c 3T3 cells, showed an induction of VEGF transcription, mRNA synthesis, and protein secretion as defined by transcriptional reporter, Northern blot, and ELISA assays. The FGF-2–induced VEGF expression was not dependent on select matrix adherence or signaling components because VEGF mRNA expression induced by FGF-2 was equally activated on serum, basement membrane, and fibronectin matrix substrates. Systemic application of anti-VEGF antibodies significantly repressed FGF-2–induced angiogenesis over control antibody by 88% (p < 0.001). These data support an FGF-2 angiogenic model that is dependent on endothelial cell activation, stromal cell infiltration, and VEGF expression by the infiltrating stromal cell population.

Hypoxia-mediated regulation of gene expression in mammalian cells

The molecular mechanism underlying oxygen sensing in mammalian cells has been extensively investigated in the areas of glucose transport, glycolysis, erythropoiesis, angiogenesis and catecholamine metabolism. Expression of functionally operative representative proteins in these specific areas, such as the glucose transporter 1, glycolytic enzymes, erythropoietin, vascular endothelial growth factor and tyrosine hydroxylase are all induced by hypoxia. Recent studies demonstrated that both transcriptional activation and post‐transcriptional mechanisms are important to the hypoxia‐mediated regulation of gene expression. In this article, the cis‐acting elements and trans‐acting factors involved in the transcriptional activation of gene expression will be reviewed. In addition, the mechanisms of post‐transcriptional mRNA stabilization will also be addressed. We will discuss whether these two processes of regulation of hypoxia‐responsive genes are mechanistically linked and co‐operative in nature.

N-Acetyl-Cysteine Promotes Angiostatin Production and Vascular Collapse in an Orthotopic Model of Breast Cancer

The antioxidant N-acetyl-cysteine (NAC) has been shown to be chemopreventive in clinical studies, and in recent studies, has shown promise in preventing tumor progression. Although the effects of NAC on tumorigenesis have been associated with decreased angiogenesis, the mechanism of the anti-angiogenic activity has not been determined. In the following study, we describe a novel mechanism whereby NAC therapy blocks MDA-MB-435 breast carcinoma cell proliferation and metastasis in an in vivo tumorigenic model. Athymic nude mice bearing MDA-MB-435 xenografts were treated with systemic NAC daily for 8 weeks. NAC treatment resulted in endothelial cell apoptosis and reduction of microvascular density within the core of the tumor leading to significant tumor cell apoptosis/necrosis. Angiostatin accumulated in tumors from NAC-treated but not control animals. Additional studies using a vascular endothelial growth factor-dependent chicken chorioallantoic membrane angiogenic assay recapitulated NAC-induced endothelial apoptosis and coordinate production of angiostatin, a potent endothelial apoptotic factor. In vitro studies showed angiostatin was formed in endothelial cultures in a vascular endothelial growth factor- and NAC-dependent manner, a process that requires endothelial cell surface plasminogen activation. These results suggest that systemic NAC therapy promotes anti-angiogenesis through angiostatin production, resulting in endothelial apoptosis and vascular collapse in the tumor.