Differential expression of VEGFA, TIE2, and ANG2 but not ADAMTS1 in rat mesenteric microvascular arteries and veins.

Microvessels respond to metabolic stimuli (e.g. pO(2)) and hemodynamic forces (e.g. shear stress and wall stress) with structural adaptations including angiogenesis, remodeling and pruning. These responses could be mediated by differential gene expression in endothelial and smooth muscle cells. Therefore, rat mesenteric arteries and veins were excised by microsurgery, and mRNA expression of four angioadaptation-related genes was quantified by real time duplex RT-PCR in equal amounts of total RNA, correlated to two different house keeping genes (beta-actin, GAPDH). The results show higher expression of VEGFA, TIE2, and ANG2 in arteries than in veins, but equal expression of ADAMTS1. Higher availability of VEGFA mRNA in endothelial cells of arteries shown here could contribute to the maintenance of mechanically stressed blood vessels and counteract pressure-induced vasoconstriction.

RNA interference screening identifies a novel role for autocrine fibroblast growth factor signaling in neuroblastoma chemoresistance.

Chemotherapeutic drug resistance is one of the major causes for treatment failure in high-risk neuroblastoma (NB), the most common extra cranial solid tumor in children. Poor prognosis is typically associated with MYCN amplification. Here, we utilized a loss-of-function kinome-wide RNA interference screen to identify genes that cause cisplatin sensitization. We identified fibroblast growth factor receptor 2 (FGFR2) as an important determinant of cisplatin resistance. Pharmacological inhibition of FGFR2 confirmed the importance of this kinase in NB chemoresistance. Silencing of FGFR2 sensitized NB cells to cisplatin-induced apoptosis, which was regulated by the downregulation of the anti-apoptotic proteins BCL2 and BCLXL. Mechanistically, FGFR2 was shown to activate protein kinase C-δ to induce BCL2 expression. FGFR2, as well as the ligand fibroblast growth factor-2, were consistently expressed in primary NB and NB cell lines, indicating the presence of an autocrine loop. Expression analysis revealed that FGFR2 correlates with MYCN amplification and with advanced stage disease, demonstrating the clinical relevance of FGFR2 in NB. These findings suggest a novel role for FGFR2 in chemoresistance and provide a rational to combine pharmacological inhibitors against FGFR2 with chemotherapeutic agents for the treatment of NB.

Intussusceptive angiogenesis: pillars against the blood flow.

Adaptation of vascular networks to functional demands needs vessel growth, vessel regression and vascular remodelling. Biomechanical forces resulting from blood flow play a key role in these processes. It is well-known that metabolic stimuli, mechanical forces and flow patterns can affect gene expression and remodelling of vascular networks in different ways. For instance, in the sprouting type of angiogenesis related to hypoxia, there is no blood flow in the rising capillary sprout. In contrast, it has been shown that an increase of wall shear stress initiates the splitting type of angiogenesis in skeletal muscle. Otherwise, during development, both sprouting and intussusception act in parallel in building the vascular network, although with differences in spatiotemporal distribution. Thereby, in addition to regulatory molecules, flow dynamics support the patterning and remodelling of the rising vascular tree. Herewith, we present an overview of angiogenic processes with respect to intussusceptive angiogenesis as related to local haemodynamics.

An imaging spectroscopy approach for measurement of oxygen saturation and hematocrit during intravital microscopy.

OBJECTIVE: Oxygen supply and partial pressure are key determinants of tissue metabolic status and are also regulators of vascular function including production of reactive oxygen species, vascular remodeling, and angiogenesis. The objective of this study was to develop an approach for the determination of oxygen saturation and hematocrit for individual microvessels in trans- and epi-illumination intravital microscopy. METHODS: A spectral approach was used, taking advantage of the availability of commercial imaging systems that allow digital recording of intravital images at a number of predetermined wavelengths within a relatively short time. The dependence of validity and precision of saturation measurements on critical experimental variables (reference spectra, number and selection of wavelengths, exposure time, analysis area, analysis model) was evaluated. In addition, a software approach for two-dimensional analysis of images was developed. RESULTS: Exposure times per wavelength of about 200 ms and use of up to 50 wavelengths evenly spaced from 500 to 598 nm allow automatic discrimination of microvessels from tissue background (segmentation) with reliable determination of oxygen saturation (in trans- and epi-illumination) and hematocrit (in transillumination). CONCLUSIONS: The present imaging spectroscopy approach allows detailed assessment of oxygen transport and other functional parameters at the microcirculatory level.