Josef Anrather

Identification and Characterization of CD39/Vascular ATP Diphosphohydrolase

Vascular ATP diphosphohydrolase (ATPDase) is a plasma membrane-bound enzyme that hydrolyses extracellular ATP and ADP to AMP. Analysis of amino acid sequences available from various mammalian and avian ATPDases revealed their close homology with CD39, a putative B-cell activation marker. We, therefore, isolated CD39 cDNA from human endothelial cells and expressed this in COS-7 cells. CD39 was found to have both immunological identity to, and functional characteristics of, the vascular ATPDase. We also demonstrated that ATPDase could inhibit platelet aggregation in response to ADP, collagen, and thrombin, and that this activity in transfected COS-7 cells was lost following exposure to oxidative stress. ATPDase mRNA was present in human placenta, lung, skeletal muscle, kidney, and heart and was not detected in brain. Multiple RNA bands were detected with the CD39 cDNA probe that most probably represent different splicing products. Finally, we identified an unique conserved motif, DLGGASTQ, that could be crucial for nucleotide binding, activity, and/or structure of ATPDase. Because ATPDase activity is lost with endothelial cell activation, overexpression of the functional enzyme, or a truncated mutant thereof, may prevent platelet activation associated with vascular inflammation.

Regulation of NF-κB RelA Phosphorylation and Transcriptional Activity by p21 ras and Protein Kinase Cζ in Primary Endothelial Cells

The activity of the transcription factor NF-κB is thought to be regulated mainly through cytoplasmic retention by IκB molecules. Here we present evidence of a second mechanism of regulation acting on NF-κB after release from IκB. In endothelial cells this mechanism involves phosphorylation of the RelA subunit of NF-κB through a pathway involving activation of protein kinase Cζ (PKCζ) and p21 ras . We show that transcriptional activity of RelA is dependent on phosphorylation of the N-terminal Rel homology domain but not the C-terminal transactivation domain. Inhibition of phosphorylation by dominant negative mutants of PKCζ or p21 ras results in loss of RelA transcriptional activity without interfering with DNA binding. Raf/MEK, small GTPases, phosphatidylinositol 3-kinase, and stress-activated protein kinase pathways are not involved in this mechanism of regulation.

TNF-α Gene Expression in Macrophages: Regulation by NF-κB Is Independent of c-Jun or C/EBPβ

The interaction of transcription factors is critical in the regulation of gene expression. This study characterized the mechanism by which NF-κB family members interact to regulate the human TNF-α gene. A 120-bp TNF-α promoter-reporter, possessing binding sites for NF-κB (κB3), C/EBPβ (CCAAT/enhancer binding protein β), and c-Jun, was activated by cotransfection of plasmids expressing the wild-type version of each of these transcription factors. Employing adenoviral vectors, dominant-negative versions of NF-κB p65, and c-Jun, but not C/EBPβ, suppressed (p < 0.05–0.001) LPS-induced TNF-α secretion in primary human macrophages. Following LPS stimulation, NF-κB p50/p65 heterodimers bound to the κB3 site and c-Jun to the −103 AP-1 site of the TNF-α promoter. By transient transfection, NF-κB p65 and p50 synergistically activated the TNF-α promoter. In contrast, no synergy was observed between NF-κB p65, with or without NF-κB p50, and c-Jun or C/EBPβ, even in the presence of the coactivator p300. The contribution of the upstream κB binding sites was also examined. Following LPS stimulation, the κB1 site bound both NF-κB p50/p65 heterodimers and p50 homodimers. The binding by NF-κB p50 homodimers to the κB1, but not to the κB3, site contributed to the inability of macrophages to respond to a second LPS challenge. In summary, adjacent κB3 and AP-1 sites in the human TNF-α promoter contribute independently to LPS-induced activation. Although both the κB1 and κB3 sites bound transcriptionally active NF-κB p50/p65 heterodimers, only the κB1 site contributed to down-regulation by NF-κB p50 homodimers.

The intron-exon structure of the porcine E-selectin-encoding gene.

We have cloned and sequenced the gene encoding porcine E-selectin. The gene comprises 12 exons and 11 introns. Two pseudoexons are contained within intron 4 and intron 6. These sequences are similar to the corresponding exons in the human E-selectin sequence; however, they are not present in the porcine E-selectin-encoding cDNA. Transcription starts at position -498 relative to the translation initiation site. The first ATG is located within exon 2. Translation stops in exon 11 leaving exon 12 untranslated in its entirety.

Genetic engineering of endotheiial cells

Abstract: Endotheiial cell activation is a major obstacle to successful xenotransplantation. The activated phenotype is largely based on the transcriptional induction of a number of genes and their products. Due to the large number of new gene products in activated endotheiial cells, it is not feasible to target each of them individually for therapeutic intervention. A common factor important for the induction of many, if not all, genes induced upon endotheiial cell activation if NF‐KB. It is thus a reasonable target for inhibition if one is to attempt to inhibit endotheiial cell activation genetically. Donor animals for xenotransplantation are amenable to genetic manipulation and we believe that transgenic animals carrying several transgenes will be the standard of future experimental and clinical xenotransplantation.

Protective Responses of Endothelial Cells

Endothelial cells (EC) as they normally exist in their quiescent state perform critical functions in maintaining blood flow and avoiding thrombosis. Various proinflammatory stimuli can induce EC to be activated, which results in recruitment, trans-endothelial migration and activation of circulating leukocytes, procoagulation, platelet aggregation, and other responses associated with inflammation. In the case of an organ that is transplanted, these reactions associated with EC activation accompany the rejection of such organ. We suggested, several years ago, that EC activation is the underlying basis of rejection of organ xenografts, i.e., grafts such as a heart or kidney transplanted across different species. While antibodies and complement in the recipient are clearly implicated in EC activation and xenograft rejection, investigators in the 1980s showed that, under certain circumstances, grafts can survive indefinitely despite the presence of antigraft antibodies and complement. We referred to the survival of an organ in the presence of anti-organ antibodies and complement as “accommodation.” One possible mechanism that we proposed to explain accommodation of these grafts was that, under certain circumstances the EC in the graft up-regulate the expression of “protective genes” that would prevent those reactions associated with EC activation that presumably lead to rejection. We have since found that such protective genes do exist and that they can play such a role.