Development of hemodynamically relevant acquired arterio-venous fistulae in patients with venous malformations.

BACKGROUND: Venous malformations tend to retain their slow-flow behavior, even in progressive disease or regression following therapy. OBJECTIVE: The aim of this study is to analyze the development of acquired hemodynamic relevant arterio-venous fistulae in patients with slow-flow malformations. METHODS: This study is a retrospective analysis based on a consecutive local registry at a tertiary care Interdisciplinary Center for Vascular Anomalies. Patients with venous malformations and development of secondary arterio-venous fistulae were included. Indications for therapy of the vascular malformation were based on patients' symptoms and complications. The following endpoints were of clinical interest and were assessed: origin of development of arteriovenous fistula, development of secondary comorbidities as a result of the vascular malformation. For analysis we focused on descriptive statistics. RESULTS: Out of 1213 consecutive patients with vascular malformations, in 6 patients perfusion changed from slow flow to arterio-venous fast-flow patterns. Four patients developed the fistula after local trauma in the area of the malformation, the other 2 patients developed the fistula due to progression of the disease and recurrent thrombophlebitis. These 2 patients had no trauma or interventions at the time of arterio-venous fistula development. CONCLUSIONS: Acquired arterio-venous fast-flow fistula in patients with slow flow vascular malformation is very rare and might be a result of local trauma or the progression of the disease with recurrent thrombophlebitis. Specific evidence-based treatment options for these patients do not exist.

c-Ha-ras oncogene expression increases choline uptake, CTP: phosphocholine cytidylyltransferase activity and phosphatidylcholine biosynthesis in the immortalized human keratinocyte cell line HaCaT.

The effect of c-Ha-ras transfection on phosphatidylcholine biosynthesis of the keratinocyte cell line HaCaT was investigated. It was shown that ras-transfection caused a 3-fold increase of choline incorporation into phosphatidylcholine. By investigating the mechanisms underlying this phenomenon, two targets were obtained. First, the choline uptake was elevated by 2-fold in ras-transfected HaCaT cells as compared with untransfected HaCaT cells, and second, the activity of the rate-limiting enzyme of phosphatidylcholine biosynthesis, CTP:phosphocholine cytidylyltransferase, was increased by 43%. Stimulation of HaCaT cells and ras-transfected HaCaT cells with oleate revealed that the increased activity of cytidylyltransferase might be due to a higher level of enzyme. In these experiments, a 75% increase of the specific activity of fully stimulated, membrane-bound cytidylyltransferase was found in ras-transfected HaCaT cells. Choline kinase which has been previously described as a target of ras-transfection in fibroblasts was unaffected.

Growth factors stimulate phosphorylation of CTP:phosphocholine cytidylyltransferase in HeLa cells.

The effect of insulin and epidermal growth factor on the phosphorylation of CTP:phosphocholine cytidylyltransferase (EC 2.7.7.15) was investigated in HeLa cells. For the first time, cytidylyltransferase phosphorylation was shown to be influenced by growth factors in cell culture experiments. The rephosphorylation of cytidylyltransferase after an oleate-mediated dephosphorylation and translocation to membranes was increased after 2 min in the presence of insulin or epidermal growth factor by 99% and 76%, respectively, compared with controls. However, the increased phosphorylation of cytidylyltransferase did not have an effect on its subcellular distribution. Furthermore, purified cytidylyltransferase preincubated with alkaline phosphatase is a substrate for p44mapk, a member of the mitogen-activated protein (MAP) kinase family downstream of the growth factor receptors, in vitro. In accordance with the in vivo data, in vitro phosphorylation of cytidylyltransferase by p44mapk occurred after 2 min.

A selective inhibitor of cyclic AMP-dependent protein kinase, N-[2-bromocinnamyl(amino)ethyl]-5-isoquinolinesulfonamide (H-89), inhibits phosphatidylcholine biosynthesis in HeLa cells.

In this study, we report that the potent and selective inhibitor of cyclic AMP-dependent protein kinase, N-[2-bromocinnamyl(amino)ethyl]-5-isoquinolinesulfonamide (H-89) interferes with the incorporation of choline into phosphatidylcholine in HeLa cells. Treatment of cells with 10 microM H-89 for 1 h decreases the phosphatidylcholine biosynthesis by 50%. This inhibition is prevented by simultaneous addition of 10 microM forskolin, while the choline uptake itself is not affected by H-89.

Identification of a putative membrane-interacting domain of CTP:phosphocholine cytidylyltransferase from rat liver.

A putative membrane-interacting domain of CTP:phosphocholine cytidylyltransferase (CT) was identified using two peptide-specific antibodies. One antibody (SA2) was raised against the N-terminus of CT (amino acid residues 1-17) and the other antibody (SA209) against an alpha-helical domain of the enzyme (amino acid residues 247-257). Both antibodies quantitatively immunoprecipitated CT from rat liver cytosol and showed specificity towards CT when octylglucoside extracts of rat liver cytosol were assessed by Western blot analysis. However, further experiments revealed that the antibodies had different characteristics. Whereas the antibody directed against the N-terminus of CT (SA2) did not influence CT/membrane interaction, the new antibody (SA209) against the alpha-helical domain of the enzyme interfered with this interaction. Our results provide experimental evidence that the alpha-helical domain (amino acid residues 228-287) of CT may serve as a membrane-interacting domain.

The mitogen-activated protein kinases system (MAP kinase cascade): its role in skin signal transduction. A review.

Mitogen-activated protein (MAP) kinases are proline-directed kinases which are downstream components of a pathway involving p21ras and the serine/threonine kinase Raf-1. They represent an important link between the signal transduction processes at the level of the plasma membrane and the final nuclear events. Not only various growth factors and cytokines, but also other signals such as UV-light or extracellular matrix components are able to activate MAP kinases. We believe that the MAP kinase cascade may play a significant role in regulating cell proliferation and differentiation in human epidermis. In this review we summarize the rapidly increasing knowledge in this field of signal transduction and discuss some very recent results on MAP kinases and their role in skin biology.

N-[2-bromocinnamyl(amino)ethyl]-5-isoquinolinesulphonamide (H-89) inhibits incorporation of choline into phosphatidylcholine via inhibition of choline kinase and has no effect on the phosphorylation of CTP:phosphocholine cytidylyltransferase.

We have shown previously that N-[2-bromocinnamyl(amino)-ethyl]-5-isoquinolinesulphonamide (H-89), a selective inhibitor of cyclic-AMP-dependent protein kinase (PKA), inhibits phosphatidylcholine biosynthesis in HeLa cells. In the present study, we elucidated the mechanism underlying the described inhibition. Treatment of cells with 10 microM H-89 had no effect on the phosphorylation of CTP:phosphocholine cytidylyltransferase. However, H-89 slightly affected the distribution of cytidylyltransferase between cytosol and membranes, but the cellular 1,2-diacylglycerol content was not influenced. Furthermore, pulse-chase experiments revealed that H-89 did not affect cytidylyltransferase activity. Instead, H-89 inhibited choline kinase, the enzyme catalysing the first step in the CDP-choline pathway. In the presence of 10 microM H-89, choline kinase activity was inhibited by 36 +/- 7.6% in vitro. Additionally, the phosphorylation of choline to phosphocholine was inhibited by 30 +/- 3% in cell-culture experiments. This inhibitory effect could be partly prevented by simultaneous addition of 10 microM forskolin, indicating that choline kinase is regulated in part by PKA activity.

Evidence for phosphorylation of CTP:phosphocholine cytidylyltransferase by multiple proline-directed protein kinases.

Reversible phosphorylation of CTP:phosphocholine cytidylyltransferase, the rate-limiting enzyme of phosphatidylcholine biosynthesis, is thought to play a role in regulating its activity. In the present study, the hypothesis that proline-directed kinases play a major role in phosphorylating cytidylyltransferase is substantiated using a c-Ha-ras-transfected clone of the human keratinocyte cell line HaCaT. Cellular extracts from epidermal growth factor-stimulated HaCaT cells and from ras-transfected HaCaT cells phosphorylated cytidylyltransferase much stronger as compared with extracts from quiescent HaCaT cells. The tryptic phosphopeptide pattern of cytidylyltransferase phosphorylated by cell-free extracts from ras-transfected HaCaT cells was similar compared with the patterns of cytidylyltransferase phosphorylated by p44mpkmitogen-activated protein kinase and p34cdc2 kinase in vitro, whereas in the case of casein kinase II the pattern was different. Furthermore, in c-Ha-ras-transfected HaCaT cells the in vivo phosphorylation state of cytidylyltransferase was 2-fold higher as compared with untransfected HaCaT cells. This higher phosphorylation of cytidylyltransferase in the ras-transfected clone was reduced to a level below the phosphorylation of cytidylyltransferase in untransfected cells, using olomoucine, a specific inhibitor of proline-directed kinases. The reduced phosphorylation of cytidylyltransferase in olomoucine-treated cells correlated with an enhanced stimulation of enzyme activity by oleic acid.

Two new sphingomyelin analogues inhibit phosphatidylcholine biosynthesis by decreasing membrane-bound CTP: phosphocholine cytidylyltransferase levels in HaCaT cells.

The effects of two newly synthesized sphingomyelin analogues on phosphatidylcholine biosynthesis were investigated in the immortalized human keratinocyte cell line HaCaT. N-Acetyl-erythro-sphingosine-1-phosphocholine (AcSM) and N-octanoyl-erythro-sphingosine-1-phosphocholine (OcSM) inhibited the incorporation of choline into phosphatidylcholine with half-inhibitory concentrations (IC50) of 6 micrograms/ml and 10 micrograms/ml respectively. Further experiments revealed that AcSM and OcSM interfered with the translocation of the rate-limiting enzyme of phosphatidylcholine biosynthesis, CTP:phosphocholine cytidylyltransferase (EC 2.7.7.15), in HaCaT cells and inhibited cytidylyltransferase activity in vitro. Despite the fact that OcSM was a potent inhibitor of cytidylyltransferase in vitro, its effects on phosphatidylcholine biosynthesis and translocation of cytidylyltransferase in HaCaT cells were less pronounced as compared with AcSM. Finally, we showed that the comparatively strong effects of AcSM in cell culture experiments were due to the uptake of large amounts of this sphingomyelin analogue into the cells. The results presented demonstrate that the activity of cytidylyltransferase may be negatively regulated by a high ratio of choline head group-containing sphingolipids.