Below are four abstracts from the 4th Annual Conference on Arteriosclerosis, Thrombosis, and Vascular Biology (2003;23:a-1–a-83). Abstracts 22 and P150 have corrected author lines. Abstracts P59 and P66 were originally withdrawn but later accepted for publication.
Lipid Phosphate Phosphatase 1 Regulates Lysophosphatidic Acid Signaling in Platelets
Susan S Smyth, Yury Sigal, Zehra Pamuklar, UNC-Chapel Hill, Chapel Hill, NC; Yong Xu, Univeristy of Utah, Salt Lake City, UT; Glenn Prestwich, The Univeristy of Utah, salt Lake City, UT; Andrew J Morris, UNC-Chapel Hill, Chapel Hill, NC
Lysophosphatidic acid (LPA) is the prototypic member of a class of receptor active lysopholipid mediators that exert their effects by binding to the Edg family of G-protein-coupled cell surface receptors. LPA stimulates platelets, leukocytes, endothelial cells, and smooth muscle cells and may be a key mediator of both inflammatory and thrombotic responses. Several mechanism(s) for LPA production have been reported, however, little is known about the enzymes and pathways responsible for LPA inactivation. The dephosphorylated products of LPA are receptor-inactive and, therefore, degradation of LPA by cell surface lipid phosphate phosphatases (LPPs) could provide an important mechanism to modulate cellular responses to LPA. We report that human platelets contain both soluble and membrane-associated LPP activities. Intact platelets and platelet-derived microparticles catalyze the rapid dephosphorylation of LPA and, to a lesser extent, the related lipid spingoshine-1-phosphate. RNA and immunological analysis indicate that LPP1 is the predominant species in human platelets. This novel enzyme is a member of a recently identified class of hexa-helical integral membrane glycoproteins with a transmembrane topology that places the active site in the extracellular or luminal side of the membrane. Platelet activation increases the rate of LPP-mediated degradation of exogenous LPA and, correspondingly, appears to alter the subcellular localization of the enzyme by increasing its distribution at the plasma membrane. We have identified several novel non-hydrolyzable analogs of phosphatidic acid that are competitive inhibitors of LPP1. These compounds cause a significant potentiation of LPA production by agonist-activated platelets. Moreover, while they do not appear to activate the platelet LPA receptor(s), these compounds enhance platelet shape change elicited by LPA and increase LPA-induced spreading of platelets on immobilized fibrinogen. Our results suggest that LPPs act as negative regulators of LPA signaling and that LPP1 can oppose the production and/or action of LPA in platelets. In addition to playing an important role in modulating platelet responsiveness to LPA, platelet LPP1 may also regulate plasma levels of LPA.
Diet-Dependent Influence of Complement C1q Deficiency on Early Lesion Formation in LDL Receptor Knock-out Mice (LDLR−/−)
Vinay K Bhatia, Clare E Roberts, Joseph J Boyle, Imperial College, London, United Kingdom; David C Grimsditch, GlaxoSmithKline, Stevenage, United Kingdom; Yolande Sadler, Imperial College, London, United Kingdom; Martin Benson, GlaxoSmithKline, Stevenage, United Kingdom; Marina B Botto, Dorian O Haskard, Imperial College, London, United Kingdom
The role of complement in atherogenesis is not clearly understood. We explored the role of the classical pathway, using C1q deficient mice (C1qa−/−). LDLR−/− and C1qa−/− on a C57BL/6 background were crossed to give C1qaLDLR−/− double knock-outs. Atherosclerotic lesion area was quantified on proximal aortic sinus cross-sections stained with Oil Red O and Mayer’s Haematoxylin. Body weights, lipoprotein, total cholesterol and trigylceride profiles were equivalent in all groups. C1qa−/− did not develop lesions when fed an atherogenic diet (cholate-free, 0.25% cholesterol, 15% fat) for 36 weeks. In mice fed standard chow for 12 weeks, mean lesion area (MLA) was greater in C1qaLDLR−/− than LDLR−/−(0.54 + 0.07 v. 0.32 + 0.08 x 105 μm2, p=0.03, Mann Whitney). In contrast, in mice fed atherogenic diet for 12 weeks, MLA was less in C1qaLDLR−/− than in LDLR−/− (2.34 + 0.26 v. 3.42 + 0.19 x 105 μm2, p=0.01). Differences in MLA between LDLR−/− and C1qaLDLR−/− were no longer observed at 40-44 weeks, either on standard or atherogenic diet. Thus C1q deficiency has a complex effect on lesion formation, depending on diet composition and duration. The greater early lesion size in C1qLDLR−/− on a standard diet suggests a physiological role for C1q in the disposal of lipoproteins and/or dead cells. On the other hand, reduced early lesion development in C1qLDLR−/− mice fed an atherogenic diet is consistent with a proinflammatory effect of complement in the presence of increased atherogenic load.Figure
Transcriptional Regulation of the Human ABCA1 Gene by Hormone Nuclear Receptors And SREBP-1
Horng-Yuan Kan, Boston University School of Medicine, Boston, MA; Michael R Hayden, University of British Columbia, Vancouver, BC, Canada; Eleni E Zanni, Boston University School of Medicine, Boston, MA; Roshni Singaraja, LinHua Zhang, University of British Columbia, Vancouver, BC, Canada; Vassilis I Zannis, Boston University School of Medicine, Boston, MA; Dimitris Kardassis, University of Crete Medical School, Heraklion, Crete, Greece
The ABCA1 gene has multiple transcription initiation sites upstream of the first exon or following the first intron. We have performed functional analysis of the promoter and the first intron of the human ABCA1 gene and have investigated the role of hormone nuclear receptors and the sterol regulatory element binding protein-1 (SREBP-1) in ABCA1 gene transcription. Deletion analysis of the ABCA1 promoter in HepG2 cells identified positive and negative regulatory elements within the -3,000/-200 region. The activity of the full-length (-3000/+1) and different truncated ABCA1 promoter segments were induced 3- to 5-fold by the combination of 22-OH cholesterol and 9-cis retinoic acid. An 8 kb fragment containing the first intron of the ABCA1 gene was active in HepG2 cells and its activity was induced potently (over 60-fold) in HEK293 cells by different combinations of heterodimers of RXRα with LXRα and FXRα which have as their ligands 22-OH cholesterol and chenodeoxycholic acid respectively. DNaseI footprinting analysis of the 1,500/1 intron-1 region using purified SREBP-1 identified five protected regions which were designated A (175/181), B (222/241), C (602/631), D (986/999), and E (1112-1136). The 260/1 region containing only sites A and B was sufficient to confer induction by SREBP-1 whereas point mutations in either site A or site B reduced both the basal and the SREBP-1 induced promoter activity by approximately 50%. SREBP-1 had the opposite effect on the ABCA1 gene promoter since it repressed 30-50% the activity of various promoter fragments extending up to -3,000 relative to the transcription initiation site of the gene. Our findings suggest an important role of the intron-1 promoter on the basal and the induced expression of the ABCA1 gene in response to sterols and ligands that activate hormone nuclear receptors.
Human Heat Shock Protein 60-Induced Activation of CD4+CD28null T Cells and Acute Coronary Syndromes
B Zal, JC Kaski, Q Xu, D Cole, JP Akiyiu, G Arno, St George’s Hospital Medical School, University of London, UK; M Whelan, N Russell, Oxyvax Ltd, St George’s Hospital Medical School, University of London, UK; IA Dodi, JA Madrigal, Anthony Nolan Bone Marrow Research Centre, Royal Free and University College London, University of London, UK; C Baboonian, St George’s Hospital Medical School, University of London, UK
Acute coronary syndromes (ACS), namely unstable angina (UA) and myocardial infarction are manifestations of atherosclerotic vascular disease usually triggered by sudden rupture of a vulnerable plaque. Multiple factors determine plaque vulnerability, of which immune-inflammatory pathways are proven to be critical. Activated T cells and macrophages are major components of these pathways and are abundantly present in advanced vulnerable lesions and ruptured plaques. It is established that patients with UA, but not chronic stable angina (SA), experience a peripheral burst of an unusual sub-set of CD4+ T cells lacking the expression of CD28 marker. These CD4+CD28null cells, which expand to approximately 50% of the total CD4+ T cell compartment, are different to normal CD4+ T cells phenotypically and functionally. CD4+CD28null cells express high levels of perforin, and INF-gamma, exhibiting active cytotoxicity and cytokine-induced matrix metalloproteinases induction, leading to plaque matrix cap degradation and rupture. These features and the finding that CD4+ CD28null T cells could only be isolated from ruptured unstable plaques, suggest that they are the dominant T cells directly involved in events leading to plaque destabilisation and ACS. Triggers of activation and expansion of CD4+ CD28null cells have remained unclear despite extensive investigations, although current opinion increasingly points to stimulation by a specific antigen. We reveal the nature of the triggering antigen and demonstrate that these T cells from patients with UA (n=20), but not those with SA (n=12), specifically react with human heat shock protein 60 (hHSP60). Following stimulation with this protein, CD4+ CD28null cells exhibit a rapid and significant increase in the transcription levels of perforin and INF-gamma, compared with other antigens or unstimulated cells We also confirm that hHSP60 is MHC Class II restricted as demonstrated in antibody blocking experiments. We propose that the emergence and expansion of CD4+ CD28null cells in response to chronic in vivo stimulation by hHSP60 may mark the clinical turning point for stable atherosclerotic plaques and highlights the beginning of a process leading to plaque destabilisation, rupture, thrombosis and ACS.