Browsing by Author "Freedman, NJ"
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Item Open Access Aging-related atherosclerosis is exacerbated by arterial expression of tumor necrosis factor receptor-1: evidence from mouse models and human association studies.(Human molecular genetics, 2010-07) Zhang, L; Connelly, JJ; Peppel, K; Brian, L; Shah, SH; Nelson, S; Crosslin, DR; Wang, T; Allen, A; Kraus, WE; Gregory, SG; Hauser, ER; Freedman, NJAging is believed to be among the most important contributors to atherosclerosis, through mechanisms that remain largely obscure. Serum levels of tumor necrosis factor (TNF) rise with aging and have been correlated with the incidence of myocardial infarction. We therefore sought to determine whether genetic variation in the TNF receptor-1 gene (TNFR1) contributes to aging-related atherosclerosis in humans and whether Tnfr1 expression aggravates aging-related atherosclerosis in mice. With 1330 subjects from a coronary angiography database, we performed a case-control association study of coronary artery disease (CAD) with 16 TNFR1 single-nucleotide polymorphisms (SNPs). Two TNFR1 SNPs significantly associated with CAD in subjects >55 years old, and this association was supported by analysis of a set of 759 independent CAD cases. In multiple linear regression analysis, accounting for TNFR1 SNP rs4149573 significantly altered the relationship between aging and CAD index among 1811 subjects from the coronary angiography database. To confirm that TNFR1 contributes to aging-dependent atherosclerosis, we grafted carotid arteries from 18- and 2-month-old wild-type (WT) and Tnfr1(-/-) mice into congenic apolipoprotein E-deficient (Apoe(-/-)) mice and harvested grafts from 1 to 7 weeks post-operatively. Aged WT arteries developed accelerated atherosclerosis associated with enhanced TNFR1 expression, enhanced macrophage recruitment, reduced smooth muscle cell proliferation and collagen content, augmented apoptosis and plaque hemorrhage. In contrast, aged Tnfr1(-/-) arteries developed atherosclerosis that was indistinguishable from that in young Tnfr1(-/-) arteries and significantly less than that observed in aged WT arteries. We conclude that TNFR1 polymorphisms associate with aging-related CAD in humans, and TNFR1 contributes to aging-dependent atherosclerosis in mice.Item Open Access Anti–β1-adrenergic receptor antibodies and heart failure: causation, not just correlation(Journal of Clinical Investigation, 2004-05-15) Freedman, NJ; Lefkowitz, Robert JItem Open Access Human umbilical cord blood endothelial progenitor cells decrease vein graft neointimal hyperplasia in SCID mice.(Atherosclerosis, 2010-09) Zhu, S; Malhotra, A; Zhang, L; Deng, S; Zhang, T; Freedman, NJ; Storms, R; Peppel, K; Goldschmidt Clermont, PJ; Dong, COBJECTIVE: Vein graft endothelial damage is a key step in the development of neointimal hyperplasia, leading to vein graft failure. We sought to determine whether exogenous endothelial progenitor cells could promote vein graft re-endothelialization, and thereby ameliorate neointimal hyperplasia. RESULTS: Carotid artery interposition grafting was performed with syngeneic inferior vena cavae in mice with severe combined immunodeficiency (SCID). Lineage-negative human umbilical cord blood (hUCB) cells (or medium alone) were injected into vein-grafted mice intra-operatively and 2 weeks post-operatively. In vein grafts from hUCB cell-injected mice, we found human HLA-expressing endothelial cells, as well as increased levels of VEGF and FGF-2. Furthermore, hUCB cells secreted VEGF and FGF-2 in vitro. The markedly enhanced endothelial regeneration, likely resulting from both direct engraftment and paracrine actions of hUCB cells, inhibited inflammatory response, diminished intimal cell proliferation, and reduced neointimal hyperplasia in the vein grafts. CONCLUSIONS: hUCB cells may accelerate vein graft re-endothelialization via both direct differentiation into endothelial cells and release of paracrine factors to enhance endothelial regeneration and reduce inflammation. These data highlight a potential therapeutic role for cellular therapy in vessel injury.Item Open Access Human umbilical cord blood-derived endothelial cells reendothelialize vein grafts and prevent thrombosis.(Arteriosclerosis, thrombosis, and vascular biology, 2010-11) Brown, MA; Zhang, L; Levering, VW; Wu, JH; Satterwhite, LL; Brian, L; Freedman, NJ; Truskey, GAOBJECTIVE: To accelerate vein graft reendothelialization and reduce vein graft thrombosis by infusing human umbilical cord blood-derived endothelial cells (hCB-ECs) because loss of endothelium contributes to vein graft thrombosis and neointimal hyperplasia. RESULTS: Under steady flow conditions in vitro, hCB-ECs adhered to smooth muscle cells 2.5 to 13 times more than ECs derived from peripheral blood or human aorta (PItem Open Access Monoclonal antibodies reveal receptor specificity among G-protein-coupled receptor kinases.(Proc Natl Acad Sci U S A, 1996-07-23) Oppermann, M; Diversé-Pierluissi, M; Drazner, MH; Dyer, SL; Freedman, NJ; Peppel, KC; Lefkowitz, RJGuanine nucleotide-binding regulatory protein (G protein)-coupled receptor kinases (GRKs) constitute a family of serine/threonine kinases that play a major role in the agonist-induced phosphorylation and desensitization of G-protein-coupled receptors. Herein we describe the generation of monoclonal antibodies (mAbs) that specifically react with GRK2 and GRK3 or with GRK4, GRK5, and GRK6. They are used in several different receptor systems to identify the kinases that are responsible for receptor phosphorylation and desensitization. The ability of these reagents to inhibit GRK- mediated receptor phosphorylation is demonstrated in permeabilized 293 cells that overexpress individual GRKs and the type 1A angiotensin II receptor. We also use this approach to identify the endogenous GRKs that are responsible for the agonist-induced phosphorylation of epitope-tagged beta2- adrenergic receptors (beta2ARs) overexpressed in rabbit ventricular myocytes that are infected with a recombinant adenovirus. In these myocytes, anti-GRK2/3 mAbs inhibit isoproterenol-induced receptor phosphorylation by 77%, while GRK4-6-specific mAbs have no effect. Consistent with the operation of a betaAR kinase-mediated mechanism, GRK2 is identified by immunoblot analysis as well as in a functional assay as the predominant GRK expressed in these cells. Microinjection of GRK2/3-specific mAbs into chicken sensory neurons, which have been shown to express a GRK3-like protein, abolishes desensitization of the alpha2AR-mediated calcium current inhibition. The intracellular inhibition of endogenous GRKs by mAbs represents a novel approach to the study of receptor specificities among GRKs that should be widely applicable to many G-protein-coupled receptors.Item Open Access Neuropeptide Y gene polymorphisms confer risk of early-onset atherosclerosis.(PLoS Genet, 2009-01) Shah, SH; Freedman, NJ; Zhang, L; Crosslin, DR; Stone, DH; Haynes, C; Johnson, J; Nelson, S; Wang, L; Connelly, JJ; Muehlbauer, M; Ginsburg, GS; Crossman, DC; Jones, CJ; Vance, J; Sketch, MH; Granger, CB; Newgard, CB; Gregory, SG; Goldschmidt Clermont, PJ; Kraus, WE; Hauser, ERNeuropeptide Y (NPY) is a strong candidate gene for coronary artery disease (CAD). We have previously identified genetic linkage to familial CAD in the genomic region of NPY. We performed follow-up genetic, biostatistical, and functional analysis of NPY in early-onset CAD. In familial CAD (GENECARD, N = 420 families), we found increased microsatellite linkage to chromosome 7p14 (OSA LOD = 4.2, p = 0.004) in 97 earliest age-of-onset families. Tagged NPY SNPs demonstrated linkage to CAD of a 6-SNP block (LOD = 1.58-2.72), family-based association of this block with CAD (p = 0.02), and stronger linkage to CAD in the earliest age-of-onset families. Association of this 6-SNP block with CAD was validated in: (a) 556 non-familial early-onset CAD cases and 256 controls (OR 1.46-1.65, p = 0.01-0.05), showing stronger association in youngest cases (OR 1.84-2.20, p = 0.0004-0.09); and (b) GENECARD probands versus non-familial controls (OR 1.79-2.06, p = 0.003-0.02). A promoter SNP (rs16147) within this 6-SNP block was associated with higher plasma NPY levels (p = 0.04). To assess a causal role of NPY in atherosclerosis, we applied the NPY1-receptor-antagonist BIBP-3226 adventitially to endothelium-denuded carotid arteries of apolipoprotein E-deficient mice; treatment reduced atherosclerotic neointimal area by 50% (p = 0.03). Thus, NPY variants associate with atherosclerosis in two independent datasets (with strong age-of-onset effects) and show allele-specific expression with NPY levels, while NPY receptor antagonism reduces atherosclerosis in mice. We conclude that NPY contributes to atherosclerosis pathogenesis.Item Open Access Structural basis for receptor subtype-specific regulation revealed by a chimeric beta 3/beta 2-adrenergic receptor.(Proc Natl Acad Sci U S A, 1993-04-15) Liggett, SB; Freedman, NJ; Schwinn, DA; Lefkowitz, RJThe physiological significance of multiple G-protein-coupled receptor subtypes, such as the beta-adrenergic receptors (beta ARs), remains obscure, since in many cases several subtypes activate the same effector and utilize the same physiological agonists. We inspected the deduced amino acid sequences of the beta AR subtypes for variations in the determinants for agonist regulation as a potential basis for subtype differentiation. Whereas the beta 2AR has a C terminus containing 11 serine and threonine residues representing potential sites for beta AR kinase phosphorylation, which mediates rapid agonist-promoted desensitization, only 3 serines are present in the comparable region of the beta 3AR, and they are in a nonfavorable context. The beta 3AR also lacks sequence homology in regions which are important for agonist-mediated sequestration and down-regulation of the beta 2AR, although such determinants are less well defined. We therefore tested the idea that the agonist-induced regulatory properties of the two receptors might differ by expressing both subtypes in CHW cells and exposing them to the agonist isoproterenol. The beta 3AR did not display short-term agonist-promoted functional desensitization or sequestration, or long-term down-regulation. To assign a structural basis for these subtype-specific differences in agonist regulation, we constructed a chimeric beta 3/beta 2AR which comprised the beta 3AR up to proline-365 of the cytoplasmic tail and the C terminus of the beta 2AR. When cells expressing this chimeric beta 3/beta 2AR were exposed to isoproterenol, functional desensitization was observed. Whole-cell phosphorylation studies showed that the beta 2AR displayed agonist-dependent phosphorylation, but no such phosphorylation could be demonstrated with the beta 3AR, even when beta AR kinase was overexpressed. In contrast, the chimeric beta 3/beta 2AR did display agonist-dependent phosphorylation, consistent with its functional desensitization. In addition to conferring functional desensitization and phosphorylation to the beta 3AR, the C-terminal tail of the beta 2AR also conferred agonist-promoted sequestration and long-term receptor down-regulation.