Molecular profiling of angiogenesis markers

Abstract

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.

Figure 1.

Real-time quantitative RT-PCR quantitation of gene expression in mouse TRAMP tumors. Total RNA and cDNA was prepared from two normal prostate (Normal 1 and Normal 2) and two TRAMP tumors (TRAMP 1 and TRAMP 2). The level of gene expression in the cDNA was measured in the presence of its respective primer and SYBR Green I Dye in real-time quantitative PCR. PCR reactions for each sample were performed in duplicate for both target gene and cyclophilin normalizer. The level of gene expression was calculated after normalizing against the cyclophilin level in each sample and presented as relative units in the graph.

Figure 2.

Induction of neovascularization in nude mouse skin by SK-MEL-2 human melanoma VEGF-transfectants. Human SK-MEL-2 melanoma cells were transfected with human VEGF₁₆₅ under the direction of a CMV promoter and clones (hVEGF-Low and hVEGF-High) were isolated. A: Northern blot detection of VEGF expression in hVEGF-Low and hVEGF-High clones. The fold increase of hVEGF-High over hVEGF-Low was calculated after normalization to the β-actin. B: Real-time quantitative RT-PCR detection of absolute VEGF copy numbers in VEGF-Low and VEGF-High clones. C: Angiogenesis in nude mouse skin was induced by subdermal injection of 0.25cc Matrigel containing 1.5 × 10⁶ transfected cells as indicated or no cells (control). Tissue was harvested and photographed at day 6.

Figure 3.

Real-time quantitative RT-PCR quantitation of mRNA copy number in skin specimens from nude mice injected with Matrigel +/− hVEGF-transfectants. On dissection, Matrigel was removed and total RNA and cDNA was prepared from the overlying skin associated with control Matrigel, Matrigel + hVEGF-Low transfectants, and Matrigel + hVEGF-High transfectants. The precisely measured and 10-fold serially diluted cDNAs templates (Ang-1, Ang-2, Flt-1, KDR, PECAM, VE-cadherin, Tie-1, and Tie-2) were used as external standard curve. The copy numbers of each mRNA was calculated after normalizing to million copies of cyclophilin in each sample and data are presented as copy number/million cyclophilin mRNA molecules.