Suppression of vascular endothelial growth factor-mediated endothelial cell protection by survivin targeting.

The protective genes that mediate endothelial cell (EC) survival during angiogenesis have not been completely characterized. Here, we show that an antisense oligonucleotide to the apoptosis inhibitor survivin suppressed de novo expression of survivin in ECs by vascular endothelial cell growth factor (VEGF). In contrast, the survivin antisense oligonucleotide did not affect anti-apoptotic bcl-2 levels in endothelium.

Interleukin-11 up-regulates survivin expression in endothelial cells through a signal transducer and activator of transcription-3 pathway.

Interleukin-11 (IL-11) reduces injury both in vivo and in vitro, but the mechanisms are unknown. Stimulation of serum- and growth factor-deprived HUVEC with IL-11 increased survivin mRNA and protein expression levels in a dose-dependent manner, with maximal induction at 50 to 100 ng/ml of IL-11. Survivin mRNA expression peaked after 3 to 6 hours of IL-11 treatment and decreased by 24 hours.

Human vascular endothelial cells stimulate a lower frequency of alloreactive CD8+ pre-CTL and induce less clonal expansion than matching B lymphoblastoid cells: development of a novel limiting dilution analysis method based on CFSE labeling of lymphocytes.

We have previously shown that human endothelial cells (EC) are less efficient than professional APC, e.g., B lymphoblastoid cells (BLC), at stimulating allogeneic CD8(+) T cells to develop into CTL.

Tumor necrosis factor alpha increases human cerebral endothelial cell Gb3 and sensitivity to Shiga toxin.

Hemolytic uremic syndrome (HUS) is associated with intestinal infection by enterohemorrhagic Escherichia coli strains that produce Shiga toxins. Globotriaosylceramide (Gb3) is the functional receptor for Shiga toxin, and tumor necrosis factor alpha (TNF-alpha) upregulates Gb3 in both human macrovascular umbilical vein endothelial cells and human microvascular brain endothelial cells. TNF-alpha treatment enhanced Shiga toxin binding and sensitivity to toxin.

Caveolin-1 associates with TRAF2 to form a complex that is recruited to tumor necrosis factor receptors.

Tumor necrosis factor (TNF) receptor-associated factor (TRAF) 2 is an intracellular adapter protein, which, upon TNF stimulation, is directly recruited to the intracellular region of TNF receptor 2 (TNFR2) or indirectly, via TRADD, to the intracellular region of TNF receptor 1 (TNFR1). In cultured human umbilical vein endothelial cells, endogenous TRAF2 colocalizes with the membrane-organizing protein caveolin-1 at regions of enrichment subjacent to the plasma membrane as detected by confocal fluorescence microscopy. Both endogenous and transfected TRAF2 protein coimmunoprecipitate with caveolin-1 in the absence of ligand.

Selective inhibition of NF-kappaB activation by a peptide that blocks the interaction of NEMO with the IkappaB kinase complex.

Activation of the transcription factor nuclear factor (NF)-kappaB by proinflammatory stimuli leads to increased expression of genes involved in inflammation. Activation of NF-kappaB requires the activity of an inhibitor of kappaB (IkappaB)-kinase (IKK) complex containing two kinases (IKKalpha and IKKbeta) and the regulatory protein NEMO (NF-kappaB essential modifier). An amino-terminal alpha-helical region of NEMO associated with a carboxyl-terminal segment of IKKalpha and IKKbeta that we term the NEMO-binding domain (NBD).

The death domain of tumor necrosis factor receptor 1 is necessary but not sufficient for Golgi retention of the receptor and mediates receptor desensitization.

TNF signals are mediated through two different receptors, TNFR1 and TNFR2. In endothelial cells, TNFR1 is predominantly localized in the Golgi apparatus and TNFR2 on the plasma membrane. To investigate structural features responsible for the disparate localization, endothelial cells were transfected with epitope-tagged or green fluorescent protein-fused wild type and mutant receptor molecules.

In vivo formation of complex microvessels lined by human endothelial cells in an immunodeficient mouse.

We have identified conditions for forming cultured human umbilical vein endothelial cells (HUVEC) into tubes within a three-dimensional gel that on implantation into immunoincompetent mice undergo remodeling into complex microvessels lined by human endothelium. HUVEC suspended in mixed collagen/fibronectin gels organize into cords with early lumena by 24 h and then apoptose. Twenty-hour constructs, s.

Human TNF can induce nonspecific inflammatory and human immune-mediated microvascular injury of pig skin xenografts in immunodeficient mouse hosts.

TNF activates endothelial cells to express cell surface molecules that are necessary to recruit a local infiltrate of leukocytes. Because the actions of this proinflammatory cytokine are not species restricted, we investigated whether human TNF can up-regulate porcine endothelial adhesion molecules to elicit human T cell infiltration and damage of pig skin xenografts in a chimeric immunodeficient mouse model. We have previously demonstrated the vigorous rejection of human skin allografts and the absence of injury to porcine skin xenografts in human PBMC-SCID/beige mice.

Human vascular endothelial cells stimulate memory but not naive CD8+ T cells to differentiate into CTL retaining an early activation phenotype.

Endothelial cell (EC)-selective alloreactive CTL may mediate alloimmune vascular injury. In the present study, EC-selective CTL were generated in cocultures of purified human CD8+ T cells with allogeneic EC and were compared with conventional CTL against corresponding B lymphoblastoid cells (BLC). EC caused activation and expansion of memory but not naive CD8+ T cells, which differentiated into EC-selective CTL that retained high surface expression of CD69, CD25, and CD62L and displayed low intracellular perforin content.

Cytoprotection of human umbilical vein endothelial cells against apoptosis and CTL-mediated lysis provided by caspase-resistant Bcl-2 without alterations in growth or activation responses.

Graft endothelial cells are primary targets of host CTL-mediated injury in acute allograft rejection. As an in vitro trial of gene therapy to reduce CTL-mediated endothelial injury, we stably transduced early passage HUVEC with a caspase-resistant mutant form (D34A) of the anti-apoptotic gene Bcl-2. Bcl-2 transductants were compared with HUVEC transduced in parallel with an enhanced green fluorescent protein (EGFP) gene.

A phosphatidylinositol 3-kinase/Akt pathway, activated by tumor necrosis factor or interleukin-1, inhibits apoptosis but does not activate NFkappaB in human endothelial cells.

Tumor necrosis factor (TNF) and interleukin-1 (IL-1) activate the transcription of both anti-apoptotic and pro-inflammatory gene products in human endothelial cells (EC) via NFkappaB. Here we report that both TNF and IL-1 activate the anti-apoptotic protein kinase Akt in growth factor and serum-deprived EC, assessed by Western blotting for phospho-Akt. Phosphorylation of Akt is blocked by LY294002 or wortmannin, inhibitors of phosphatidylinositol 3-kinase (PI 3-kinase).

IL-11 activates human endothelial cells to resist immune-mediated injury.

IL-11, a gp130-signaling cytokine, is protective in several in vivo models of immune-mediated and inflammatory injury. HUVECs express IL-11 receptor alpha-chain and gp130. Human IL-11 causes rapid (2-10 min) tyrosine phosphorylation of gp130.

Large-scale culture and selective maturation of human Langerhans cells from granulocyte colony-stimulating factor-mobilized CD34+ progenitors.

Dendritic cells (DCs) play a critical role as APCs in the induction of the primary immune response. Their capacity for Ag processing and presentation is tightly regulated, controlled by a terminal developmental sequence accompanied by striking changes in morphology, organization, and function. The maturation process, which converts DCs from cells adapted for Ag accumulation to cells adapted for T cell stimulation, remains poorly understood due in part to difficulties in the culture and manipulation of DCs of defined lineages.

Control of apoptosis during angiogenesis by survivin expression in endothelial cells.

Mechanisms controlling endothelial cell survival during angiogenesis were investigated. Stimulation of quiescent endothelial cells with mitogens, including vascular endothelial growth factor and basic fibroblast growth factor, induced up to approximately 16-fold up-regulation of the cell cycle-regulated apoptosis inhibitor survivin. Mitogen stimulation rapidly increased survivin RNA expression in endothelial cells, which peaked after 6 to 10 hours in culture and decreased by 24 hours.

Interferon-gamma elicits arteriosclerosis in the absence of leukocytes.

Atherosclerosis and post-transplant graft arteriosclerosis are both characterized by expansion of the arterial intima as a result of the infiltration of mononuclear leukocytes, the proliferation of vascular smooth muscle cells (VSMCs) and the accumulation of extracellular matrix. They are also associated with the presence of the immunomodulatory cytokine interferon-gamma (IFN-gamma). Moreover, in mouse models of atheroma formation or allogeneic transplantation, the serological neutralization or genetic absence of IFN-gamma markedly reduces the extent of intimal expansion.

Recent advances in the molecular basis of TNF signal transduction.

Substantial progress has been made in the last decade toward defining the signaling pathways that can be activated by TNF and identifying the relevant intracellular signaling molecules. The in vivo consequences of targeted disruption of many of the genes encoding proteins involved in TNF signaling (as discussed in this review) are quite different from those observed for knockout mutations of TNF and the TNF receptors (Erickson et al, 1994; Marino et al, 1997; Rothe et al, 1993) that use these molecules. This suggests that there is still much to be learned about the mechanisms for determining specificity in signaling.

Immunobiology of human vascular endothelium.

The author's laboratory studies interactions between human T lymphocytes and vascular endothelial cells (EC). Our work is organized around three hypotheses. First, we propose that vascular EC can initiate secondary (i.

Human endothelial cells augment early CD40 ligand expression in activated CD4+ T cells through LFA-3-mediated stabilization of mRNA.

Human endothelial cells (EC) augment CD40 ligand (CD40L) expression on PHA-activated CD4+ T cells at early times (e.g., 4-6 h).