New blood vessel formation requires the coordination of endothelial cell division and the morphogenetic movements of vessel expansion, but it is not known how this integration occurs. Here, we show that endothelial cells regulate division orientation during the earliest stages of blood vessel formation, in response to morphogenetic cues. In embryonic stem (ES) cell-derived vessels that do not experience flow, the plane of endothelial cytokinesis was oriented perpendicular to the vessel long axis. We also demonstrated regulated cleavage orientation in vivo, in flow-exposed forming retinal vessels. Daughter nuclei moved away from the cleavage plane after division, suggesting that regulation of endothelial division orientation effectively extends vessel length in these developing vascular beds. A gainof-function mutation in VEGF signaling increased randomization of endothelial division orientation, and this effect was rescued by a transgene, indicating that regulation of division orientation is a novel mechanism whereby VEGF signaling affects vessel morphogenesis. Thus, our findings show that endothelial cell division and morphogenesis are integrated in developing vessels by flow-independent mechanisms that involve VEGF signaling, and this cross talk is likely to be critical to proper vessel morphogenesis. IntroductionBlood vessels form and expand in both development and disease, via processes that include vasculogenesis, angiogenesis, and intussusception (reviewed in Risau, 1 Eichmann et al, 2 Coultas et al 3 ). Sprouting angiogenesis is the coordinated migration of groups of endothelial cells from vessels and their subsequent fusion to form new interconnections. In this way, simple vascular tubes are ramified and extended to form a primitive vascular plexus. This vessel plexus forms at numerous sites in the embryo, including the yolk sac, the head mesenchyme, and surrounding the neural tube. The primitive vascular plexus is then remodeled under the influence of blood flow and interactions with mural cells. Thus, the initial pattern of vessels serves as a template for remodeling that leads to a mature vasculature.During formation of the primitive vascular plexus, several cellular processes must be regulated and integrated. Specifically, endothelial cells respond to some morphogenetic cues by sprouting, while actively dividing to expand the pool of endothelial cells. One level of integration occurs via the signaling pathways that promote angiogenesis, because many affect both endothelial cell division and morphogenesis. The VEGF signaling pathway is an example of this mode of integration, because it regulates both cell division and branching morphogenesis (reviewed in Rousseau et al, 4 Kliche and Waltenberger, 5 Ferrara et al,6 Nagy and Senger, 7 and Shibuya and Claesson-Welsh 8 ). VEGF-A (VEGF) binds 2 highaffinity receptors on endothelial cells, flk-1 (VEGFR-2) and flt-1 (VEGFR-1), and perturbation of VEGF signaling by genetic deletion of either receptor affects both endothelial cell division and morphogenesis. ...

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Primary RPE cells and those from cell lines had different responses to medium-term culture or hypoxic stress. Primary isolation of hfRPE cells with careful control of culture conditions to assure adequate differentiation is recommended when using this cell as an example of a highly polarized epithelium. For disease, use of RPE cells that do not require long-term culture are more efficient and may be more relevant to study certain pathologies.

Aims-To determine if erythromycin given from birth reduces the inflammatory response and the incidence and severity of chronic lung disease. Methods-Seventy five infants less than 30 weeks of gestation and ventilated from birth for lung disease were randomly assigned to receive erythromycin intravenously for 7 days or to no treatment. Ureaplasma urealyticum was detected in tracheal secretions by culture and polymerase chain reaction. DiVerential cell counts were obtained from bronchoalveolar lavage fluid collected daily for 5 days and concentrations of the cytokines interluekins IL-1 and IL-8, and tumour necrosis factor (TNF-) were measured. Chronic lung disease (CLD) was defined as oxygen dependency at 36 weeks of gestation. Results-Nine infants (13%) were positive for U urealyticum. The inflammatory cytokines in the lungs increased over the first 5 days of life in all babies, but no association was found between their concentrations and the development of CLD. Those treated with erythromycin showed no significant diVerences from the nontreated group in the diVerential cell counts or concentrations of the cytokines. The two groups had a similar incidence of CLD. Babies infected with U urealyticum did not have a more pronounced cytokine response than those without infection. Chorioamnionitis was associated with significantly higher concentrations of IL-1 and IL-8 on admission: these babies had less severe acute lung disease and developed significantly less CLD. Conclusions-U urealyticum in the trachea was not associated with an increased inflammatory response in preterm infants. Erythromycin did not reduce the incidence or severity of CLD. (Arch Dis Child Fetal Neonatal Ed 1998;78:F10-F14)

Identifying noninvasive markers of pulmonary inflammation would be useful in assessing new therapies in children. Breath condensate is a simple and potentially acceptable sample medium even in small children. The technique has previously been used in adults, but not children with cystic fibrosis.The technique was assessed in 36 children with cystic fibrosis (mean age 10.4 yrs) and 17 control subjects, analysing samples for nitrite, interleukin(IL)-8 and salivary and nasal contamination. Correlations were made between levels of the inflammatory markers and forced expiratory volume in one second/forced vital capacity, chest radiograph score and use of inhaled steroids.On samples without significant contamination (<10 u . L -1 amylase) nitrite was detected in 93% of samples at a median concentration of 3.0 mM compared with 50% of control samples at a median of 0.5 mM. Condensate amylase levels did not correlate with the nitrite value obtained (r=0.31). IL-8 was detected in 33% of CF samples.Breath condensate is an acceptable method of sample collection in children. Nitrite was raised in breath condensate from patients with cystic fibrosis when compared with control subjects. Eur Respir J 2000; 15: 955±957. Pulmonary inflammation in children with cystic fibrosis (CF) begins soon after birth [1], and arresting this progressive inflammation is central to CF research. It would be desirable therefore to identify a measure of pulmonary inflammation (single or combined), that is more sensitive than pulmonary function testing, noninvasive, repeatable and applicable to young children. The assessment of exhaled breath in CF has not been fruitful: nitric oxide levels are normal [2], or reduced [3] in CF when compared with control subjects, and exhaled pentane is raised in CF, but prone to gastrointestinal contamination [4].The measurement of inflammatory mediators in breath condensate (exhaled aerosolized bronchoalveolar lining fluid), might provide a noninvasive, direct assessment of pulmonary inflammation. Condensate is capable of carrying molecules <65 kDa [5]: that could include the majority of pro-inflammatory pulmonary cytokines that are increased in CF. Nitrite This technique, however, has not been applied to children with CF, and the aim of the current study was therefore to evaluate the reliability and acceptability of this technique, assessing whether children with CF have raised nitrite (like their adult counterparts [6]), and to assess for the first time whether IL-8, a dominant pro-inflammatory cytokine in CF [8], is found in measurable quantities in exhaled condensate. MethodsThe study was performed at the authors' regional paediatric CF centre (Edinburgh, Scotland, UK). Control samples were collected from children who were either staff relatives or attending fracture clinic with single stable fractures and no history of chronic respiratory illness. Children with CF were clinically stable at the time of assessment.The equipment for condensate collection has previously been used successfully in adults, ...