Prolactin upregulates sphingosine kinase-1 expression and activity in the human breast cancer cell line MCF7 and triggers enhanced proliferation and migration

Sphingosine kinases (SK) catalyze the formation of sphingosine-1-phosphate (S1P) which plays a crucial role in cell growth and survival. Here, we show that prolactin (PRL) biphasically activates the SK-1, but not the SK-2 subtype, in the breast adenocarcinoma cell-line MCF7. A first peak occurs after minutes of stimulation and is followed by a second delayed activation after hours of stimulation. A similar biphasic effect on SK-1 activity is seen for 17beta-estradiol (E(2)). The delayed activation of SK-1 derives from an upregulated mRNA and protein expression and is due to increased SK-1 promoter activity and mechanistically involves STAT5 activation as well as protein kinase C and the classical mitogen-activated protein kinases. Furthermore, glucocorticoids also block both hormone-induced SK-1 expression and activity. Functionally, long-term stimulation of MCF7 cells with PRL or E(2) is well known to trigger increased cell proliferation and migration. Both hormone-induced cell responses critically involve SK-1 activation since the depletion of SK-1, but not SK-2, by siRNA transfection abolishes the hormone-induced cell proliferation and migration. In summary, our data show that PRL and E(2) cause a pronounced delayed SK-1 activation which is due to increased gene transcription, and critically determines the capability of cells to grow and move. Thus, the SK-1 may represent a novel attractive target for anti-tumor therapy.

Sphingosylphosphorylcholine acts in an anti-inflammatory manner in renal mesangial cells by reducing interleukin-1beta-induced prostaglandin E2 formation

Sphingosylphosphorylcholine (SPC) is a bioactive lipid that binds to G protein-coupled-receptors and activates various signaling cascades. Here, we show that in renal mesangial cells, SPC not only activates various protein kinase cascades but also activates Smad proteins, which are classical members of the transforming growth factor-beta (TGFbeta) signaling pathway. Consequently, SPC is able to mimic TGFbeta-mediated cell responses, such as an anti-inflammatory and a profibrotic response. Interleukin-1beta-stimulated prostaglandin E(2) formation is dose-dependently suppressed by SPC, which is paralleled by reduced secretory phospholipase A(2) (sPLA(2)) protein expression and activity. This effect is due to a reduction of sPLA(2) mRNA expression caused by inhibited sPLA(2) promoter activity. Furthermore, SPC upregulates the profibrotic connective tissue growth factor (CTGF) protein and mRNA expression. Blocking TGFbeta signaling by a TGFbeta receptor kinase inhibitor causes an inhibition of SPC-stimulated Smad activation and reverses both the negative effect of SPC on sPLA(2) expression and the positive effect on CTGF expression. In summary, our data show that SPC, by mimicking TGFbeta, leads to a suppression of proinflammatory mediator production and stimulates a profibrotic cell response that is often the end point of an anti-inflammatory reaction. Thus, targeting SPC receptors may represent a novel therapeutic strategy to cope with inflammatory diseases.

Protein kinase C alpha-dependent phosphorylation of the mRNA-stabilizing factor HuR: implications for posttranscriptional regulation of cyclooxygenase-2

In this study, we investigated the molecular mechanisms underlying the ATP analogue adenosine-5'-O-(3-thio)triphosphate-induced nucleocytoplasmic shuttling of the mRNA stabilizing factor HuR in human (h) mesangial cells (MC). Using synthetic protein kinase C (PKC) inhibitors and small interfering RNA approaches, we demonstrated that knockdown of PKC alpha efficiently blocked the ATP-dependent nuclear HuR export to the cytoplasm. The functional importance of PKC alpha in HuR shuttling is highlighted by the high cytosolic HuR content detected in hMC stably overexpressing PKC alpha compared with mock-transfected cells. The ATP-induced recruitment of HuR to the cytoplasm is preceded by a direct interaction of PKC alpha with nuclear HuR and accompanied by increased Ser phosphorylation as demonstrated by coimmunoprecipitation experiments. Mapping of putative PKC target sites identified serines 158 and 221 as being indispensable for HuR phosphorylation by PKC alpha. RNA pull-down assay and RNA electrophoretic mobility shift assay demonstrated that the HuR shuttling by ATP is accompanied by an increased HuR binding to cyclooxygenase (COX)-2 mRNA. Physiologically, the ATP-dependent increase in RNA binding is linked with an augmentation in COX-2 mRNA stability and subsequent increase in prostaglandin E(2) synthesis. Regulation of HuR via PKC alpha-dependent phosphorylation emphasizes the importance of posttranslational modification for stimulus-dependent HuR shuttling.

No temporal association between influenza outbreaks and invasive pneumococcal infections

OBJECTIVE: To assess whether the influenza peak in populations precedes the annual peak for invasive pneumococcal infections (IPI) in winter. DESIGN: Ecological study. Active surveillance data on influenza A and IPI in children up to 16 years of age collected from 1997 to 2003 were analysed. SETTING: Paediatric hospitals in Germany. Patients: Children under 16 years of age. RESULTS: In all years under study, the influenza A season did not appear to affect the IPI season (p = 0.49). Specifically, the influenza peak never preceded the IPI peak. CONCLUSION: On a population level there was no indication that the annual influenza epidemic triggered the winter increase in the IPI rate or the peak of the IPI distribution in children.

rFVIIa in the treatment of persistent hemorrhage in pediatric patients on ECMO following surgery for congenital heart disease

BACKGROUND: Patients who require extracorporeal membrane oxygenation (ECMO) postsurgery for congenital heart disease (CHD) frequently experience severe bleeding episodes. Whereas recombinant-activated factor VII (rFVIIa) has proven efficacy in counteracting intractable hemorrhage in various scenarios, its use in patients on ECMO is limited by the increased risk for thrombotic events. METHODS: Between December 2004 and January 2006, ECMO was used in 10 pediatric patients following cardiac surgery, of whom seven were treated with rFVIIa because of intractable hemorrhage. Their medical records were reviewed with respect to variations in chest tube output and transfusion requirements, occlusion of or thrombus formation in the ECMO circuit and the occurrence of thromboembolic events. Outcome and rate of ECMO circuit occlusion were compared with historic controls. RESULTS: Three patients died, and four survived (none of the deaths was attributable to thrombus formation or bleeding). All patients were treated with aprotinin prior to and during rFVIIa therapy. Two patients developed an occlusion of the oxygenator, one after receiving co-medication with a FXIII concentrate, another after RBC transfusion in the ECMO system. In two patients, thrombus formation was observed in the ECMO system on inspection after discontinuation. Thromboembolic events were not observed. CONCLUSIONS: Recombinant-activated factor VII in a median dosage of 90 microg.kg(-1) was used in seven pediatric patients on ECMO. Rates of ECMO system occlusions and mortality did not differ from historic controls. Neither the reduction of chest tube output nor the blood product transfusion requirements did reach statistical significance.

Hippocampal functional connectivity reflects verbal episodic memory network integrity

Using functional magnetic resonance imaging during a verbal memory task, we investigated correlations of signal fluctuations within the hippocampus and ipsilateral frontal as well as temporal areas in temporal lobe epilepsy patients. Declarative memory abilities were additionally examined before and after temporal lobe epilepsy surgery. A significant difference exists in functional connectivity between patients whose mnemonic functions deteriorated and those who remained stable or improved. Univariate analyses showed significantly higher preoperative coupling between the hippocampus and Brodmann area 22 for the group that decreased in verbal learning. We suggest greater coupling to reflect higher functional network integrity. Postoperatively reduced learning ability in patients with higher preoperative coupling underlines the importance of hippocampal interaction with cortical areas for successful memory formation.

Experimental ovine model of primary venous valve incompetence: a pilot study

PURPOSE: To compare two techniques used to create a larger animal model of venous valve incompetence. MATERIALS AND METHODS: To achieve vein dilatation as the primary cause of valve incompetence, common carotid jugular vein (JV) fistulas were created and optional filters were placed into the JV of sheep. Altogether, nine inferior vena cava filters were placed in three sheep in two stages. Six filters were placed caudal to the most caudal JV valve in three sheep and removed 6 weeks later. Then, three filters were placed across the most caudal valve in two sheep with competent valves and removed 3 weeks later. A common carotid artery-JV fistula was created in three sheep and followed-up for 1-3 weeks. Ascending and descending venograms were obtained to determine the JV sizes and function of their valves. The JVs removed at necropsy were studied with venoscopy. RESULTS: Only one of the six JVs with filters caudal to the most caudal valve had incompetent valves after filter removal at 6 weeks. In addition, only one of three JVs with the filter across the valve had incompetent valves after filter removal at 3 weeks. At 1-3-week follow-up of the group with common carotid artery-JV fistula, all three JVs had incompetent valves in the cephalad vein portion, but only one JV had an incompetent valve in its caudal portion. At venoscopy, the incompetent valves showed various degrees of damage ranging from shortening to the destruction of valve leaflets. CONCLUSION: Dilation of the valve annulus with a removable vena cava filter failed to produce valve incompetence. The promising results with the common carotid artery-JV fistula justify further detailed research.

Percutaneous autologous venous valve transplantation: short-term feasibility study in an ovine model

BACKGROUND: Limited experience with bioprosthetic venous valve percutaneously inserted into femoral veins in 15 patients has been promising in short-term results only to show disappointing long-term results. Percutaneous autogenous venous valve (PAVV) transplantation was explored in an ovine model as a possible alternative treatment. METHODS: PAVV consisted of a vein segment containing a valve that was attached to a stent template. The stent templates (n = 9) were designed and hand made in our research laboratory. They consist of two stainless steel square stents 13 or 15 mm in diameter to fit the ovine jugular veins (JV), which ranges from 10 to 15 mm in diameter. A valve-containing segment of JV was harvested and attached with sutures and barbs inside the stent template (n = 9). The valve devices were then manually folded and front loaded inside the 4 cm chamber of the 13F delivery sheath and delivered into the contralateral JV by femoral vein approach. Transplanted PAVVs were studied by immediate and 3 months venograms. Animals were euthanized at 3 months, and jugular veins harvested to perform angioscopic evaluations in vitro. RESULTS: PAVV transplantation was successful in all nine animals. Good valve function with no reflux was observed on immediate and 3 months venograms in eight valves. The transplanted maximal JV diameter ranged from 10.2 mm to 15.4 mm (mean 13.1 +/- 1.5 mm). Venoscopic examination revealed intact, flexible, nonthickened valve leaflets in eight specimens. One PAVV exhibited normal function of one leaflet only; the other cusp was accidentally cut during the transplantation procedure. All transplanted autologous valves were free of thrombus and incorporated into the vein wall of the host vessel. CONCLUSION: This study demonstrated that autogenous valve transplants remained patent and competent without long-term anticoagulation for up to 3 months. The percutaneous autogenous venous valve may provide in future minimally invasive treatment for patients with chronic deep venous insufficiency, but long-term studies need to be done to document its continued patency and function.

Percutaneous vein occlusion with small intestinal submucosa: an experimental pilot study in Swine and sheep

PURPOSE: The objective of this study was to investigate the feasibility, outcomes, and amount of small intestinal submucosa (SIS) material needed for embolization of jugular vein (JV) in a swine and sheep model. Our hypothesis was that SIS would cause vein occlusion. MATERIALS AND METHODS: The external JVs (EJV) in swine (n = 6) and JVs in sheep (n = 6) were occluded with SIS fan-folded compressed strips. After percutaneous puncture of the peripheral portion of the EJV or JV, a TIPS set was used to exit their lumen centrally through the skin. The SIS strips were delivered into the isolated venous segment with a pull-through technique via a 10-Fr sheath. Follow-up venograms were done immediately after placement and at the time of sacrifice at 1 or 3 months. Gross examinations focused on the EJV or JV and their surrounding structures. Specimens were evaluated by histology. RESULTS: SIS strip(s) placement was successful in all cases, with immediate vein occlusion seen in 23 of 24 veins (95.8%). All EJVs treated with two strips and all JVs treated with three or four strips remained closed on 1- and 3-month follow-up venograms. Two EJVs treated with one strip and one JV treated with two strips were partially patent on venograms at 1 and 3 months. There has been one skin inflammatory reaction. Necropsies revealed excluded EJV or JV segments with SIS incorporation into the vein wall. Histology demonstrated various stages of SIS remodeling with fibrocytes, fibroblasts, endothelial cells, capillaries, and inflammatory cells. CONCLUSION: We conclude that EJV and JV ablation with SIS strips using percutaneous exit catheterization is feasible and effective in animal models. Further exploration of SIS as vein ablation material is recommended.

Angioscopy for experimental evaluation of optional IVC filters

PURPOSE: To demonstrate the feasibility of direct angioscopic visualization of an optional inferior vena cava (IVC) filter in situ and during retrieval. MATERIALS AND METHODS: Angioscopy was used for direct visualization of optional IVC filters in six sheep. Cavograms were obtained before the filters were retrieved. After successful filter retrieval, segmental IVC perfusion was performed to evaluate filter retrieval-related damage to the IVC wall. Therefore, all branch vessels were ligated before the IVC segment was flushed with normal saline solution until it was fully distended. Then, the inflow was terminated and the IVC segment observed for deflation. Subsequently, the IVC was harvested en bloc, dissected, and inspected macroscopically. RESULTS: The visibility of IVC filters at angioscopy was excellent. During the retrieval procedure, filter collapse and retraction into the sheath were clearly demonstrated. Angioscopy provided additional information to that obtained with cavography, demonstrating adherent material in three filters. Three filters in place for more than 2 months could not be retrieved because the filter legs were incorporated into the IVC wall. After filter retrieval, there was no perforation at segmental IVC perfusion. At macroscopic inspection of the IVC lumen, a small piece of detached endothelium was found in one animal. CONCLUSION: Angioscopy enabled the direct evaluation of optional IVC filters in situ and during retrieval. Compared with cavography, angioscopy provided additional information about the filter in situ and the retrieval procedure. Future applications of this technique could include studies of filter migration, compression, and clot-trapping efficacy.

Percutaneous technique for creation of tricuspid regurgitation in an ovine model

Experimental models of tricuspid regurgitation (TR) are needed to study the percutaneous placement of prosthetic atrioventricular valves. The purpose of this study was to develop an appropriate simple and reproducible percutaneous experimental model for creation of tricuspid regurgitation. Tricuspid regurgitation was successfully created through papillary muscle avulsion using a guide-wire loop in seven sheep with regurgitation documented on right ventricular angiograms and a significant increase in heart rate and right atrial pressures. Acute onset of tricuspid regurgitation was poorly tolerated in one animal that died. Autopsy examinations showed avulsion of one papillary muscle in four animals and two papillary muscles in three animals.