Browsing by Subject "Receptors, Lipoprotein"
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Item Dissecting the Molecular Basis of the Antiphospholipid Syndrome(2008-05-13) Ramesh, Sangeetha; Shaul, P. W.The antiphospholipid syndrome (APS) is an autoimmune disorder characterized by circulating antiphospholipid antibodies (aPL), thrombotic events, recurrent pregnancy loss, and increased risk of coronary artery disease. The endothelium is a critical direct target of aPL, which cause increases in adhesion molecule expression and procoagulant activity. However, the molecular mechanisms underlying aPL actions on endothelium are unknown. Nitric oxide (NO) produced by endothelial NO synthase (eNOS) prevents leukocyte adhesion and thrombosis. In the present study we determined if aPL-induced alterations in endothelial cell phenotype are mediated by aPL actions on eNOS. Normal human IgG (NHIgG) and human IgG containing polyclonal aPL were obtained from healthy individuals and APS patients, respectively, and purified by protein G-sepharose chromatography. We found that aPL prevents VEGF-mediated attenuation of monocyte adhesion to cultured endothelial cells and this is reversed by an NO donor, indicating a role for eNOS antagonism. In contrast, NHIgG has no effect on adhesion. Whereas NHIgG does not alter eNOS activation, stimulation of eNOS by VEGF and other agonists is fully antagonized by aPL. In intact mice, NO-dependent, acetylcholine-induced increases in carotid vascular conductance are unchanged by NHIgG treatment but impaired following aPL, indicating that these processes are operative in vivo. Additional studies in cultured endothelial cells demonstrated that aPL attenuates eNOS activation by inhibiting Ser1179 phosphorylation. We further found that deprivation of the cell surface protein beta 2 glycoprotein I (beta 2GPI) from endothelial cells prevents aPL inhibition of eNOS, and that FC1, a monoclonal antibody against beta 2GPI, mimics the effects of aPL. Receptor-associated protein or RAP, an antagonist of the LDL receptor (LDLR) family, fully prevents aPL antagonism of eNOS in endothelial cells. Moreover, eNOS antagonism by aPL as indicated in vivo in carotid vascular conductance studies is absent in apoER2 -/- mice. Thus, aPL-induced changes in endothelial cell phenotype are mediated by eNOS antagonism, which is due to impaired Ser1179 phosphorylation and requires beta 2GPI and apoER2. eNOS antagonism through these mechanisms represent a novel, potentially critical proximal process in the pathogenesis of thrombosis and cardiovascular disease in APS.Item Low-Density Lipoprotein Receptors in Signaling Modulation and Development(2010-05-14) Dietrich, Martin Frederik; Herz, JoachimThe Low-Density Lipoprotein Receptor gene family is a group of ancient membrane receptors. Originally implied in cargo transport and development of atherosclerosis, the number of members and the diversity of functions have been greatly expanded. LRP1, LRP1b and LRP4 are gene family members that are implicated in the regulation of signaling pathways at the intracellular, extracellular and transcriptional level. These regulations confer viability, control the cellular proliferation at several molecular steps, and allow for proper organ formation by moderating and integrating cellular signaling pathways. / The use of knockin mutant mice has, for the first time, implicated the extracellular domains of LRP1b and LRP4 in signaling modulation in development. While the complete knockout of either receptor is embryonically lethal, the expression of a truncated receptor, spanning only the extracellular domain, confers viability and only a mitigated phenotype. / For LRP4, the difference is most visible in the kidney. The present LRP4 extracellular preserves thresholds critical for organogenesis, yet, the complete absence displays a subpenetrant phenotype of kidney agenesis. In this thesis work, results demonstrate the ability of the LRP4 extracellular domain to not only bind a broad variety of soluble ligands in the extracellular space, but further to influence the Wnt, and possibly others, signaling pathways that are required for kidney development. / In an osteoblast-specific model of LRP1 knock-out, the relationship between theLRP1and the PDGF receptor has been further investigated. LRP1 is known to negatively regulate the PDGF receptor. However, the exact mechanism(s) are not fully understood. In the wild-type, PDGF receptor beta binds directly to LRP1 upon ligand stimulation. LRP1 knockout leads to significant upregulation of the PDGF receptor beta at the protein level. The stimulation of the receptor with PDGF-BB, its corresponding ligand, leads to overactivation of the signaling pathway with both increased turnover and phosphorylation/activation of the receptor, demonstrated by cellular proliferation and p21 downregulation. In vivo, the LRP1 knockout leads to a bone-derived hyperproliferation with formation of tumors at the epiphysis. The in vitro experiments are supporting evidence, combined with previously published literature, to imply the LRP1/PDGF receptor pathway.