Tissue factor (TF), a transmembrane receptor for coagulation factor VII͞VIIa, is aberrantly expressed in human cancers. We demonstrated a significant correlation between TF and vascular endothelial growth factor (VEGF) production in 13 human malignant melanoma cell lines (r 2 ؍ 0.869, P < 0.0001). Two of these cell lines, RPMI-7951, a high TF and VEGF producer, and WM-115, a low TF and VEGF producer, were grown s.c. in severe combined immunodeficient mice. The high-producer cell line generated solid tumors characterized by intense vascularity, whereas the low producer generated relatively avascular tumors, as determined by immunohistologic staining of tumor vascular endothelial cells with anti-von Willebrand factor antibody. To investigate the structure-function relationship of TF and VEGF, a lowproducer melanoma cell line (HT144) was transfected with a TF cDNA containing the full-length sequence, a cytoplasmic deletion mutant lacking the coding sequence for the distal three serine residues (potential substrates for protein kinase C), or an extracellular domain mutant, which has markedly diminished function for activation of factor X. Cells transfected with the full-length sequence produced increased levels of both TF and VEGF. Transfectants with the full-length sequence and the extracellular domain mutant produced approximately equal levels of VEGF mRNA. However, cells transfected with the cytoplasmic deletion mutant construct produced increased levels of TF, but little or no VEGF. Thus, the cytoplasmic tail of TF plays a role in the regulation of VEGF expression in some tumor cells.
Objective. To study the intracellular mechanism involved in the up-regulation of tissue factor (TF) on endothelial cells (ECs) by antiphospholipid antibodies (aPL), we examined the effects of aPL on the transcription, expression, and function of TF, the expression of interleukin-6 (IL-6) and IL-8, the induction of inducible nitric oxide synthase (iNOS), and the phosphorylation of p38 MAPK on human umbilical vein ECs (HUVECs).Methods. Cultured HUVECs were treated with IgG aPL (from patients with antiphospholipid syndrome [APS]) or with control IgG (from normal human serum). Phorbol myristate acetate (PMA) and bacterial lipopolysaccharide (LPS) were used as positive controls. TF expression was determined on the surface of HUVECs using an enzyme-linked immunosorbent assay (ELISA). TF activity was determined with the use of a chromogenic assay in cell lysates, and TF messenger RNA (mRNA) was determined by real-time quantitative polymerase chain reaction. Phosphorylation of p38 MAPK and induction of iNOS were determined by Western blotting, and levels of IL-6 and IL-8 were determined by ELISA.Results. PMA, LPS, and aPL significantly increased the expression of TF compared with controls. This up-regulation was significantly inhibited by SB203580 (a specific inhibitor of p38 MAPK) and by MG132 (a specific inhibitor of NF-B). TF activity was significantly increased by treatment with IgG aPL and this effect was also inhibited by SB203580. Incubation of HUVECs with aPL increased TF mRNA 2-15-fold; these effects were abrogated by SB203580. IgG aPL induced significant phosphorylation of p38 MAPK and produced iNOS on HUVECs in a time-dependent manner. Treatment with IgG aPL also induced increased expression of IL-6 and IL-8 on HUVECs.Conclusion. Our data show that aPL induces significant increases in TF transcription, function, and expression, in IL-6 and IL-8 up-regulation, and in iNOS expression on HUVECs and that these processes involve phosphorylation of p38 MAPK and activation of NF-B.
The initial consult by PC achieved significant symptom improvement in patients receiving treatment in the OPC. Further prospective studies are needed.
Summary. Background: Mechanisms of thrombosis induced by antiphospholipid (aPL) antibodies include up-regulation of tissue factor (TF) expression on endothelial cells (ECs). Statins have been shown to reduce levels of TF induced by tumor necrosis factor (TNF-a) and lipopolysaccharide (LPS) on ECs. In a recent study, fluvastatin inhibited thrombogenic and proinflammatory properties of aPL antibodies in in vivo models. The aim of this study was to determine whether fluvastatin has an effect on aPL-induced expression of TF on ECs. Methods: IgGs were purified from four patients with APS (IgG-APS) and from control sera (IgG-NHS). Cultured human umbilical vein endothelial cells (HUVEC) were treated with IgG-APS or IgG-NHS or with medium alone or with phorbol myristate acetate (PMA), as a positive control. In some experiments, cells were pretreated with fluvastatin (2.5, 5 or 10 lM) with and without mevalonate (100 lM). TF expression on HUVECs was measured by ELISA. Results: PMA and the four IgG-APS preparations increased the expression of TF on EC significantly (4.9-, 2.4-, 4.2-, 3.5-and 3.1-fold, respectively), in a dose-dependent fashion. Fluvastatin (10 lM) inhibited the effects of PMA and the four IgG-APS on TF expression by 70, 47, 65, 22 and 68%, respectively, and this effect was dosedependent. Mevalonate (100 lM) completely abrogated the inhibitory effects of fluvastatin on TF expression induced by aPL. Conclusion: Because of the suggested pathogenic role of aPL on induction of TF on ECs, our data provide a rationale for using statins as a therapeutic tool in treatment of thrombosis in APS.
PVT or PVS and HVOO are known causes of graft and patient loss after pediatric liver transplantation. Increased incidences of these complications have been reported in partial livers including DDSLT or LDLT. From 1997 to 2008, 241 consecutive pediatric patients received 271 hepatic grafts at a single center. Median follow-up is 1856 days. Surgical technique, demographics, lab values, and radiologic imaging procedures were obtained utilizing OTTR to evaluate the relationship of portal and hepatic complications with risk factors, patient and graft survival. Grafts were composed of 115/271 (42.4%) partial livers of which 90 (33.2%) were DDSLT and 25 (9.2%) LDLT. Of 271 patients, 156 (57.6%) received whole-sized grafts. There were six PVC in five patients with one patient requiring retransplantation (0.34%) and no patient deaths. Utilizing all three hepatic vein orifices on the recipient hepatic vena cava and the donor hepatic vein cut short enables a wide hepatic outflow tract unlikely to twist. None of the 241 patients developed early or late complications of the hepatic vein. None of the last 128 consecutive patients who received 144 grafts over seven and a half yr have developed either early or late complications of the hepatic or portal vein. Partial-graft actuarial survival was similar to whole-graft survival (87.2% vs. 85.3% at one yr; 76.6% vs. 80.2 at three yr; p = 0.488). Likewise, patient survival was similar between partial grafts and whole grafts (93.8% vs. 93.1% at one yr; 89.8% vs. 87.2% at three yr; p = 0.688) with median follow-up of 1822 (+/-1334) days. Patients receiving partial livers were significantly younger and smaller than patients receiving whole livers (p < 0.001). Portal and hepatic venous complications may have negative effects on patient or graft survival after pediatric liver transplantation. In our series, there was one graft and no patient loss related to portal or hepatic venous complications after pediatric liver transplantation over 12 yr.
Vascular cell adhesion molecule (VCAM)-1 expression may be coupled to redox-sensitive regulatory pathways, and iron may play a role in generation of reactive oxygen species that participate in these signaling pathways. To investigate the role of iron in TNF alpha-induced VCAM-1 gene expression, human dermal microvascular endothelial cells (HDMEC) were stimulated with TNF alpha in the presence of iron chelators and examined for expression of VCAM-1. The iron chelators dipyridyl (DP) and desferoxamine (DFO) inhibited VCAM-1 protein and mRNA induction in a concentration- and time-dependent manner. The induction of VCAM-1 was not inhibited by nonmetal binding reactive oxygen species (ROS) scavengers, implying a direct effect of iron in the expression of these adhesion molecules. The effect of iron was mediated at the level of gene transcription since pretreatment with DP abrogated the TNF alpha-mediated up-regulation of VCAM-1 heterogeneous nuclear RNA. Pretreatment of HDMEC with DP prior to TNFalpha treatment had no effect on p65 nuclear localization, DNA binding, or serine phosphorylation. DP pretreatment inhibited TNF alpha- and IFN gamma-mediated interferon regulatory factor 1 (IRF-1) protein expression, although restoration of IRF-1 expression failed to reconstitute VCAM-1 expression. DP treatment also blocked VCAM-1 induction in human umbilical vein endothelium and blocked induction of a host of NF-kB activated genes in HDMEC including ICAM-1, IL-8, and tissue factor. I kappa B alpha, an NF-kappa B inducible and constitutively accessible gene not requiring chromatin remodeling for transcription, was not affected by DP treatment. These data suggest that iron plays a critical role in TNF alpha mediated VCAM-1 induction in HDMEC, and the target for iron effects may be IRF-1, NF-kappa B, and potentially chromatin remodeling.
Liver retransplantation is routinely offered at our institution. Previous reports document that patient and graft survival is significantly less after pediatric rLT compared to primary LT. This has engendered intense debate regarding optimal allocation of organs. Here, we examine our program's approach to pediatric hepatic retransplantation related to patient factors affecting outcomes. Between 1997 and 2009, 272 LTs were performed in 234 patients (mean survival 1994 +/- 1367 days) at our center. Thirty-four patients required rLT including 10 who received their primary transplant elsewhere and four who required two retransplantations. Patient survival did not differ significantly between rLT and LT at one and three yr (p = 0.56). Graft survival between rLT and LT was also similar (p = 0.606) at one and three yr. No significant difference in graft or patient survival was noted between: Patients retransplanted <30 days after LT vs. those >30 days (p = 0.152); patients transplanted with technical variants vs. whole grafts (p = 0.966); technical variants utilized for LT vs. rLT (p = 0.713); rLT recipient age (< or >5 yr; p = 0.298); or ABOI for rLT and LT (p = 0.650). Retransplantation should be offered to optimize pediatric recipient survival after LT and offers similar survival as primary transplant.
Children transplanted for acute liver failure (ALF) urgently require an optimal graft. Lower post-transplant survival compared to children transplanted for chronic liver disease. Over 10 years, 33 consecutive children transplanted for ALF were followed. Demographics, encephalopathy, intubation, dialysis, laboratory values, graft type (ABO incompatible grafts (ABOI), Large for size grafts(XL)(GRWR>5%),deceased donor segmental liver transplantation(DDSLT), living donor liver transplantation (LDLT) and whole liver transplant (WLT) were evaluated. Complications and survival were determined. ALF accounted for 33/201 (16.4%) of transplants during this period. 12/33 received ABOI, 5 XL grafts, 18 DDSLT, and 3 LDLT. Waiting time pre-transplant was 2.1 days. 1 and 3 year patient survival ALF group was 93% and 93% and graft survivals were 93 and 78.6%. Median follow-up was 1452 days. ABOI one and three year patient and graft survival in the ALF was 92 and 75%. No difference in graft or patient survival was noted in the ALF and chronic liver disease group nor the ABOI and the ABO compatible group. A combination of ABO incompatible donor livers, large for size grafts, DDSLT, LDLT and WLT led to a short wait time and subsequent graft and patient survival not significantly different than that for non-acute liver disease.
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