Three reactive compartments in venous malformations.

Vascular malformations affect 3% of neonates. Venous malformations (VMs) are the largest group representing more than 50% of cases. In hereditary forms of VMs gene mutations have been identified, but for the large group of spontaneous forms the primary cause and downstream dysregulated genes are unknown. We have performed a global comparison of gene expression in slow-flow VMs and normal saphenous veins using human whole genome micro-arrays. Genes of interest were validated with qRT-PCR. Gene expression in the tunica media was studied after laser micro-dissection of small pieces of tissue. Protein expression in endothelial cells (ECs) was studied with antibodies. We detected 511 genes more than four-fold down- and 112 genes more than four-fold up-regulated. Notably, chemokines, growth factors, transcription factors and regulators of extra-cellular matrix (ECM) turnover were regulated. We observed activation and "arterialization" of ECs of the VM proper, whereas ECs of vasa vasorum exhibited up-regulation of inflammation markers. In the tunica media, an altered ECM turnover and composition was found. Our studies demonstrate dysregulated gene expression in tunica interna, media and externa of VMs, and show that each of the three layers represents a reactive compartment. The dysregulated genes may serve as therapeutic targets.

Sub-second calcium coupling between outside medium and subplasmalemmal stores during overstimulation/depolarisation-induced ciliary beat reversal in Paramecium cells.

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 (> or =30ms) smaller than one ciliary beat ( approximately 50ms) and beyond. Particularly under overstimulation conditions ( approximately 10(-5)M Ca(2+) before, 0.5mM Ca(2+) during stimulation) we find in cilia a [Ca] peak at approximately 80ms and its decay to near-basal levels within 110ms (90%) to 170ms (100% decay). This [Ca] wave is followed, with little delay, by a [Ca] wave into subplasmalemmal Ca-stores (alveolar sacs), culminating at approximately 100ms, with a decay to original levels within 170ms. 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.

Altered expression patterns of EphrinB2 and EphB2 in human umbilical vessels and congenital venous malformations.

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.