Vascular malformations cause discomfort and pain in children and are often associated with skeletal hypertrophy. Their molecular basis is poorly understood. Ephrin ligands and Eph receptor tyrosine kinases are involved in embryonic vascular development. In mice, some ephrin/Eph family members show a complementary expression pattern in blood vessels, with ephrinB2 being expressed on arterial and EphB4 on venous endothelium. Targeted deletions of the genes reveal their essential roles for conduit vessel development in mice, suggesting similar functions during human vascular development and deregulation in vascular malformations. Here, we have defined the expression patterns of human ephrinB2, EphB4, and EphB2 in normal vessels of neonates (i.e. umbilici) and adults and compared them with those in congenital venous malformations. In adults, normal vessels of the skin, muscle, and legs express ephrinB2 and EphB2 on arterial endothelial cells (ECs), whereas EphB4 is found in arteries and veins. In the umbilicus, EphB2 is a specific marker of arterial ECs, whereas ephrinB2 is additionally expressed in venous ECs, suggesting an arterial function of the veins. In venous malformations, the expression of EphB4 is not altered, but both ephrinB2 and EphB2 are ectopically expressed in venous ECs. This may reflect a nonphysiologic arterialization of malformed veins. Our study shows that the arterial markers ephrin B2 and EphB2 are expressed in a subset of veins, and it remains to be studied whether this is cause or consequence of an altered vascular identity. (Pediatr Res 57: 537-544, 2005) Abbreviations EC, endothelial cell SAM, sterile ␣ motif ␣-SMA, ␣-smooth muscle actin SMC, smooth muscle cell During embryogenesis, blood vessels form via two distinct processes, vasculogenesis and angiogenesis (1-5). Vasculogenesis involves the de novo development of endothelial cells (ECs) from mesodermal precursors, the angioblasts, and leads to the formation of the early vascular plexus. Later, blood vessels arise mainly from the preexisting ones by sprouting, splitting, and intussusceptive growth of capillaries in a process called angiogenesis. Remodeling of the primary vascular plexus into conduit vessels and capillaries ensures perfusion of all organs and tissues with blood. In the adult, angiogenesis accounts for neovascularization accompanying cyclic reproductive changes in women, as well as pathologic processes such as tumor growth, diabetic retinopathy, and rheumatoid arthritis (6,7).To date, three growth factor families have been identified as critical players of blood vessel development: vascular endothelial growth factors, angiopoietins, and ephrins (8,9). Unlike vascular endothelial growth factors and angiopoietins, which are secreted proteins, ephrins are attached to the plasma membrane and function in cell-cell communication (10). Ephrins can be divided into two subclasses: EphrinA ligands (ephrinA1-A5) are tethered to the outer cell surface via a glycosylphosphatidylinositol anchor, whereas ephrinB ligands (ephrinB1-B3) po...

As amply documented by electrophysiology, depolarisation in Paramecium induces a Ca 2+ influx selectively via ciliary voltage-dependent Ca 2+ -channels, thus inducing ciliary beat reversal. Subsequent downregulation of ciliary Ca 2+ has remained enigmatic. We now analysed this aspect, eventually under overstimulation conditions, by quenched-flow/cryofixation, combined with electron microscope X-ray microanalysis which registers total calcium concentrations, [Ca]. This allows to follow Ca-signals within a time period (≥30 ms) smaller than one ciliary beat (∼50 ms) and beyond. Particularly under overstimulation conditions (∼10 −5 M Ca 2+ before, 0.5 mM Ca 2+ during stimulation) we find in cilia a [Ca] peak at ∼80 ms and its decay to near-basal levels within 110 ms (90%) to 170 ms (100% decay). This [Ca] wave is followed, with little delay, by a [Ca] wave into subplasmalemmal Ca-stores (alveolar sacs), culminating at ∼100 ms, with a decay to original levels within 170 ms. Also with little delay [Ca] slightly increases in the cytoplasm below. This implies rapid dissipation of Ca 2+ through the ciliary basis, paralleled by a rapid, transient uptake by, and release from cortical stores, suggesting fast exchange mechanisms to be analysed as yet. This novel type of coupling may be relevant for some phenomena described for other cells.

SummaryVascular malformations affect3 %o fn eonates.V enous malformations(VMs)are the largest group representing morethan 50% of cases. In hereditary forms of VMs gene mutations have been identified,but forthe large groupofspontaneous forms the primaryc ause and downstreamd ysregulated genesa re unknown. We have performedag lobal comparison of gene expression in slow-flow VMs and normal saphenous veins using human whole genomemicro-arrays.Genes of interest were validated with qRT-PCR.Genee xpression in the tunica media was studied after laser micro-dissection of smallp ieceso ft issue. Proteine xpression in endothelialc ells (ECs) was studied with antibodies.Wed etected5 11 genesm oret han four-foldd ownKeywords Ve nous malformation,chemokines,growth factors,transcription factors, extracellular matrix and 112g enes moret han four-foldu p-regulated. Notably, chemokines,growth factors,transcriptionfactors and regulators of extra-cellular matrix (ECM) turnoverw erer egulated. We observed activation and "arterialization" of ECs of theVM proper, whereas ECsof vasa vasorum exhibited up-regulationofinflammation markers. In the tunicam edia,ana ltered ECM turnover and composition was found.Our studies demonstratedysregulated gene expression in tunica interna, media and externa of VMs, and showt hat each of the three layers represents ar eactive compartment.Thedysregulated genesmay serveastherapeutic targets.