No abstract

Most of the antiangiogenic strategies used in oncology principally target endothelial cells through the vascular endothelial growth factor (VEGF) pathway. Multiple kinase inhibitors can secondarily reduce mural cell stabilization of the vessels by blocking platelet-derived growth factor receptor (PDGFR) activity. However, sphingosine-1-phosphate (S1P), which is also implicated in mural cell recruitment, has yet to be targeted in clinical practice. We therefore investigated the potential of a simultaneous blockade of the PDGF and S1P pathways on the chemotactic responses of vascular smooth muscle cells (VSMCs) and the resulting effects of this blockade on breast tumor growth. Due to crosstalk between the S1P and PDGF pathways, we used AG1296 and/or VPC-23019 to inhibit PDGFR-β and S1PR1/S1PR3 receptors, respectively. We showed that S1PR1 and S1PR3 are the principal receptors that mediate the S1P chemotactic signal on rat VSMCs and that they act synergistically with PDGFR-β during PDGF-B signaling. We also showed that simultaneous blockade of the PDGFR-β and S1PR1/S1PR3 signals had a synergistic effect, decreasing VSMC migration velocity toward endothelial cell and breast carcinoma cell-secreted cytokines by 65-90%. This blockade also strongly decreased the ability of VSMCs to form a three-dimensional cell network. Similar results were obtained with the combination of sunitinib malate (a VEGFR/PDGFR kinase inhibitor) and fingolimod (an S1P analog). Sunitinib malate is a clinically approved cancer treatment, whereas fingolimod is currently indicated only for treatment of multiple sclerosis. Orally administered, the combination of these drugs greatly decreased rat breast tumor growth in a syngeneic cancer model (Walker 256). This bi-therapy did not exert cumulative toxicity and histological analysis of the tumors revealed normalization of the tumor vasculature. The simultaneous blockade of these signaling pathways with sunitinib malate and fingolimod may provide an effective means of reducing tumor angiogenesis, and may improve the delivery of other chemotherapies.

In vitro angiogenesis assays are commonly used to assess pro-or anti-angiogenic drug properties. Extracellular matrix (ECM) substitutes such as Matrigel and collagen gel became very popular in in vitro 3D angiogenesis assays as they enable tubule formation by endothelial cells from culture or aortic rings. However, these assays are usually used with a single cell type, lacking the complex cellular interactions occurring during angiogenesis. Here, we report a novel angiogenesis assay using egg white as ECM substitute. We found that, similar to Matrigel, egg white elicited prevascular network formation by endothelial and/or smooth muscle cell coculture. This matrix was suitable for various cells from human, mouse, and rat origin. It is compatible with aortic ring assay and also enables vascular and tumor cell coculture. Through simple labeling (DAPI, Hoechst 33258), cell location and resulting prevascular network formation can easily be quantified. Cell transfection with green fluorescent protein improved whole cell visualization and 3D structure characterization. Finally, eggbased assay dedicated to angiogenesis studies represents a reliable and cost-effective way to produce and analyze data regarding drug effects on vascular cells.

No abstract

Improvements of microarray techniques for genotyping purposes have focused on increasing the reliability of this method. Here we report the development of a genotyping method where a microarray was spotted with stemloop probes, especially designed to optimize the hybridization specificity of complementary DNA sequences. This accurate method was used to screen for four common disease-causing mutations involved in a neurological disorder called Charcot-Marie-Tooth disease (CMT). Healthy individuals' and patients' DNA were amplified and labeled by PCR and hybridized on microarray. The spot signal intensities were 81 to 408 times greater for perfect compared with mismatched target sequences, differing by only one nucleotide (discrimination ratio) for healthy individual "homozygous" DNA. On the other hand, "heterozygous" mutant DNA samples gave rise to signal intensity ratios close to 1, as expected. The genotypes obtained by this method were perfectly consistent with those determined by direct PCR sequencing. Cross-hybridization rates were very low, resulting in further multiplexing improvements. In this study, we also demonstrated the feasibility of real-time hybridization detection of labeled synthetic oligonucleotides with concentrations as low as 2.5 nM.